Prmt5 inhibitors and uses thereof

ABSTRACT

Described herein are compounds of Formula (I), pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof. Compounds of the present invention are useful for inhibiting PRMT5 activity. Methods of using the compounds for treating PRMT5-mediated disorders are also described.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.provisional patent application, U.S. Ser. No. 61/790,525, filed Mar. 15,2013, and to U.S. provisional patent application, U.S. Ser. No.61/745,485, filed Dec. 21, 2012, the entire contents of each of whichare incorporated herein by reference.

BACKGROUND OF THE INVENTION

Epigenetic regulation of gene expression is an important biologicaldeterminant of protein production and cellular differentiation and playsa significant pathogenic role in a number of human diseases.

Epigenetic regulation involves heritable modification of geneticmaterial without changing its nucleotide sequence. Typically, epigeneticregulation is mediated by selective and reversible modification (e.g.,methylation) of DNA and proteins (e.g., histones) that control theconformational transition between transcriptionally active and inactivestates of chromatin. These covalent modifications can be controlled byenzymes such as methyltransferases (e.g., PRMT5), many of which areassociated with specific genetic alterations that can cause humandisease.

Disease-associated chromatin-modifying enzymes (e.g., PRMT5) play a rolein diseases such as proliferative disorders, metabolic disorders, andblood disorders. Thus, there is a need for the development of smallmolecules that are capable of inhibiting the activity of PRMT5.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Protein arginine methyltransferase 5 (PRMT5) catalyzes the addition oftwo methyl groups to the two w-guanidino nitrogen atoms of arginine,resulting in ω-NG, N′G symmetric dimethylation of arginine (sDMA) of thetarget protein. PRMT5 functions in the nucleus as well as in thecytoplasm, and its substrates include histones, spliceosomal proteins,transcription factors (See e.g., Sun et al., 2011, PNAS 108:20538-20543). PRMT5 generally functions as part of a molecule weightprotein complex. While the protein complexes of PRMT5 can have a varietyof components, they generally include the protein MEP50 (methylosomeprotein 50). In addition, PRMT5 acts in conjunction with cofactor SAM(S-adenosyl methionine).

PRMT5 is an attractive target for modulation given its role in theregulation of diverse biological processes. It has now been found thatcompounds described herein, and pharmaceutically acceptable salts andcompositions thereof, are effective as inhibitors of PRMT5. Suchcompounds have the general Formula (I):

or a pharmaceutically acceptable salt thereof, wherein R¹, R⁵, R⁶, R⁷,R⁸, R^(x), n, L, and Ar are as defined herein.

In some embodiments, pharmaceutical compositions are provided whichcomprise a compound described herein (e.g., a compound of Formula (I)),or a pharmaceutically acceptable salt thereof, and optionally apharmaceutically acceptable excipient.

In certain embodiments, compounds described herein inhibit activity ofPRMT5. In certain embodiments, methods of inhibiting PRMT5 are providedwhich comprise contacting PRMT5 with an effective amount of a compoundof Formula (I), or a pharmaceutically acceptable salt thereof. The PRMT5may be purified or crude, and may be present in a cell, tissue, or asubject. Thus, such methods encompass inhibition of PRMT5 activity bothin vitro and in vivo. In certain embodiments, the PRMT5 is wild-typePRMT5. In certain embodiments, the PRMT5 is overexpressed. In certainembodiments, the PRMT5 is a mutant. In certain embodiments, the PRMT5 isin a cell. In certain embodiments, the PRMT5 is in an animal, e.g., ahuman. In some embodiments, the PRMT5 is in a subject that issusceptible to normal levels of PRMT5 activity due to one or moremutations associated with a PRMT5 substrate. In some embodiments, thePRMT5 is in a subject known or identified as having abnormal PRMT5activity (e.g., overexpression). In some embodiments, a providedcompound is selective for PRMT5 over other methyltransferases. Incertain embodiments, a provided compound is at least about 10-foldselective, at least about 20-fold selective, at least about 30-foldselective, at least about 40-fold selective, at least about 50-foldselective, at least about 60-fold selective, at least about 70-foldselective, at least about 80-fold selective, at least about 90-foldselective, or at least about 100-fold selective relative to one or moreother methyltransferases.

In certain embodiments, methods of altering gene expression in a cellare provided which comprise contacting a cell with an effective amountof a compound of Formula (I), or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition thereof. In certainembodiments, the cell in culture in vitro. In certain embodiments, cellis in an animal, e.g., a human.

In certain embodiments, methods of altering transcription in a cell areprovided which comprise contacting a cell with an effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt thereof,or a pharmaceutical composition thereof. In certain embodiments, thecell in culture in vitro. In certain embodiments, the cell is in ananimal, e.g., a human.

In some embodiments, methods of treating a PRMT5-mediated disorder areprovided which comprise administering to a subject suffering from aPRMT5-mediated disorder an effective amount of a compound describedherein (e.g., a compound of Formula (I)), or a pharmaceuticallyacceptable salt thereof, or a pharmaceutical composition thereof. Incertain embodiments, the PRMT5-mediated disorder is a proliferativedisorder, a metabolic disorder, or a blood disorder. In certainembodiments, compounds described herein are useful for treating cancer.In certain embodiments, compounds described herein are useful fortreating hematopoietic cancer, lung cancer, prostate cancer, melanoma,or pancreatic cancer. In certain embodiments, compounds described hereinare useful for treating a hemoglobinopathy. In certain embodiments,compounds described herein are useful for treating sickle cell anemia.In certain embodiments, compounds described herein are useful fortreating diabetes or obesity.

Compounds described herein are also useful for the study of PRMT5 inbiological and pathological phenomena, the study of intracellular signaltransduction pathways mediated by PRMT5, and the comparative evaluationof new PRMT5 inhibitors.

This application refers to various issued patent, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference.

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., a inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in ThomasSorrell, Organic Chemistry, University Science Books, Sausalito, 1999;Smith and March, March's Advanced Organic Chemistry, 5^(th) Edition,John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various isomeric forms, e.g., enantiomers and/ordiastereomers. For example, the compounds described herein can be in theform of an individual enantiomer, diastereomer or geometric isomer, orcan be in the form of a mixture of stereoisomers, including racemicmixtures and mixtures enriched in one or more stereoisomer. Isomers canbe isolated from mixtures by methods known to those skilled in the art,including chiral high pressure liquid chromatography (HPLC) and theformation and crystallization of chiral salts; or preferred isomers canbe prepared by asymmetric syntheses. See, for example, Jacques et al.,Enantiomers, Racemates and Resolutions (Wiley Interscience, New York,1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistryof Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables ofResolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ.of Notre Dame Press, Notre Dame, Ind. 1972). The present disclosureadditionally encompasses compounds described herein as individualisomers substantially free of other isomers, and alternatively, asmixtures of various isomers.

It is to be understood that the compounds of the present invention maybe depicted as different tautomers. It should also be understood thatwhen compounds have tautomeric forms, all tautomeric forms are intendedto be included in the scope of the present invention, and the naming ofany compound described herein does not exclude any tautomer form.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds that differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of hydrogen by deuterium ortritium, replacement of ¹⁹F with ¹⁸F, or the replacement of a carbon bya ¹³C- or ¹⁴C-enriched carbon are within the scope of the disclosure.Such compounds are useful, for example, as analytical tools or probes inbiological assays.

The term “aliphatic,” as used herein, includes both saturated andunsaturated, nonaromatic, straight chain (i.e., unbranched), branched,acyclic, and cyclic (i.e., carbocyclic) hydrocarbons. In someembodiments, an aliphatic group is optionally substituted with one ormore functional groups. As will be appreciated by one of ordinary skillin the art, “aliphatic” is intended herein to include alkyl, alkenyl,alkynyl, cycloalkyl, and cycloalkenyl moieties.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C₁₋₆ alkyl” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

“Alkyl” refers to a radical of a straight-chain or branched saturatedhydrocarbon group having from 1 to 20 carbon atoms (“C₁₋₂₀ alkyl”). Insome embodiments, an alkyl group has 1 to 10 carbon atoms (“C₁₋₁₀alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms(“C₁₋₉ alkyl”). In some embodiments, an alkyl group has 1 to 8 carbonatoms (“C₁₋₈ alkyl”). In some embodiments, an alkyl group has 1 to 7carbon atoms (“C₁₋₇ alkyl”). In some embodiments, an alkyl group has 1to 6 carbon atoms (“C₁₋₆ alkyl”). In some embodiments, an alkyl grouphas 1 to 5 carbon atoms (“C₁₋₅ alkyl”). In some embodiments, an alkylgroup has 1 to 4 carbon atoms (“C₁₋₄ alkyl”). In some embodiments, analkyl group has 1 to 3 carbon atoms (“C₁₋₃ alkyl”). In some embodiments,an alkyl group has 1 to 2 carbon atoms (“C₁₋₂ alkyl”). In someembodiments, an alkyl group has 1 carbon atom (“C₁ alkyl”). In someembodiments, an alkyl group has 2 to 6 carbon atoms (“C₂₋₆ alkyl”).Examples of C₁₋₆ alkyl groups include methyl (C₁), ethyl (C₂), n-propyl(C₃), isopropyl (C₃), n-butyl (C₄), tert-butyl (C₄), sec-butyl (C₄),iso-butyl (C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl (C₅), neopentyl(C₅), 3-methyl-2-butanyl (C₅), tertiary amyl (C₅), and n-hexyl (C₆).Additional examples of alkyl groups include n-heptyl (C₇), n-octyl (C₈)and the like. In certain embodiments, each instance of an alkyl group isindependently optionally substituted, e.g., unsubstituted (an“unsubstituted alkyl”) or substituted (a “substituted alkyl”) with oneor more substituents. In certain embodiments, the alkyl group isunsubstituted C₁₋₁₀ alkyl (e.g., —CH₃). In certain embodiments, thealkyl group is substituted C₁₋₁₀ alkyl.

In some embodiments, an alkyl group is substituted with one or morehalogens. “Perhaloalkyl” is a substituted alkyl group as defined hereinwherein all of the hydrogen atoms are independently replaced by ahalogen, e.g., fluoro, bromo, chloro, or iodo. In some embodiments, thealkyl moiety has 1 to 8 carbon atoms (“C₁₋₈ perhaloalkyl”). In someembodiments, the alkyl moiety has 1 to 6 carbon atoms (“C₁₋₆perhaloalkyl”). In some embodiments, the alkyl moiety has 1 to 4 carbonatoms (“C₁₋₄ perhaloalkyl”). In some embodiments, the alkyl moiety has 1to 3 carbon atoms (“C₁₋₃ perhaloalkyl”). In some embodiments, the alkylmoiety has 1 to 2 carbon atoms (“C₁₋₂ perhaloalkyl”). In someembodiments, all of the hydrogen atoms are replaced with fluoro. In someembodiments, all of the hydrogen atoms are replaced with chloro.Examples of perhaloalkyl groups include —CF₃, —CF₂CF₃, —CF₂CF₂CF₃,—CCl₃, —CFCl₂, —CF₂Cl, and the like.

“Alkenyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 20 carbon atoms, one or morecarbon-carbon double bonds, and no triple bonds (“C₂₋₂₀ alkenyl”). Insome embodiments, an alkenyl group has 2 to 10 carbon atoms (“C₂₋₁₀alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms(“C₂₋₉ alkenyl”). In some embodiments, an alkenyl group has 2 to 8carbon atoms (“C₂₋₈ alkenyl”). In some embodiments, an alkenyl group has2 to 7 carbon atoms (“C₂₋₇ alkenyl”). In some embodiments, an alkenylgroup has 2 to 6 carbon atoms (“C₂₋₆ alkenyl”). In some embodiments, analkenyl group has 2 to 5 carbon atoms (“C₂₋₅ alkenyl”). In someembodiments, an alkenyl group has 2 to 4 carbon atoms (“C₂₋₄ alkenyl”).In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C₂₋₃alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C₂alkenyl”). The one or more carbon-carbon double bonds can be internal(such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples ofC₂₋₄ alkenyl groups include ethenyl (C₂), 1-propenyl (C₃), 2-propenyl(C₃), 1-butenyl (C₄), 2-butenyl (C₄), butadienyl (C₄), and the like.Examples of C₂₋₆ alkenyl groups include the aforementioned C₂₋₄ alkenylgroups as well as pentenyl (C₅), pentadienyl (C₅), hexenyl (C₆), and thelike. Additional examples of alkenyl include heptenyl (C₇), octenyl(C₈), octatrienyl (C₈), and the like. In certain embodiments, eachinstance of an alkenyl group is independently optionally substituted,e.g., unsubstituted (an “unsubstituted alkenyl”) or substituted (a“substituted alkenyl”) with one or more substituents. In certainembodiments, the alkenyl group is unsubstituted C₂₋₁₀ alkenyl. Incertain embodiments, the alkenyl group is substituted C₂₋₁₀ alkenyl.

“Alkynyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 20 carbon atoms, one or morecarbon-carbon triple bonds, and optionally one or more double bonds(“C₂₋₂₀ alkynyl”). In some embodiments, an alkynyl group has 2 to 10carbon atoms (“C₂₋₁₀ alkynyl”). In some embodiments, an alkynyl grouphas 2 to 9 carbon atoms (“C₂₋₉ alkynyl”). In some embodiments, analkynyl group has 2 to 8 carbon atoms (“C₂₋₈ alkynyl”). In someembodiments, an alkynyl group has 2 to 7 carbon atoms (“C₂₋₇ alkynyl”).In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C₂₋₆alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms(“C₂₋₅ alkynyl”). In some embodiments, an alkynyl group has 2 to 4carbon atoms (“C₂₋₄ alkynyl”). In some embodiments, an alkynyl group has2 to 3 carbon atoms (“C₂₋₃ alkynyl”). In some embodiments, an alkynylgroup has 2 carbon atoms (“C₂ alkynyl”). The one or more carbon-carbontriple bonds can be internal (such as in 2-butynyl) or terminal (such asin 1-butynyl). Examples of C₂₋₄ alkynyl groups include, withoutlimitation, ethynyl (C₂), 1-propynyl (C₃), 2-propynyl (C₃), 1-butynyl(C₄), 2-butynyl (C₄), and the like. Examples of C₂₋₆ alkenyl groupsinclude the aforementioned C₂₋₄ alkynyl groups as well as pentynyl (C₅),hexynyl (C₆), and the like. Additional examples of alkynyl includeheptynyl (C₇), octynyl (C₈), and the like. In certain embodiments, eachinstance of an alkynyl group is independently optionally substituted,e.g., unsubstituted (an “unsubstituted alkynyl”) or substituted (a“substituted alkynyl”) with one or more substituents. In certainembodiments, the alkynyl group is unsubstituted C₂₋₁₀ alkynyl. Incertain embodiments, the alkynyl group is substituted C₂₋₁₀ alkynyl.

“Carbocyclyl” or “carbocyclic” refers to a radical of a non-aromaticcyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C₃₋₁₀carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. Insome embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms(“C₃₋₈ carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to6 ring carbon atoms (“C₃₋₆ carbocyclyl”). In some embodiments, acarbocyclyl group has 3 to 6 ring carbon atoms (“C₃₋₆ carbocyclyl”). Insome embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms(“C₅₋₁₀ carbocyclyl”). Exemplary C₃₋₆ carbocyclyl groups include,without limitation, cyclopropyl (C₃), cyclopropenyl (C₃), cyclobutyl(C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl (C₅),cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and the like.Exemplary C₃₋₈ carbocyclyl groups include, without limitation, theaforementioned C₃₋₆ carbocyclyl groups as well as cycloheptyl (C₇),cycloheptenyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇),cyclooctyl (C₈), cyclooctenyl (C₈), bicyclo[2.2.1]heptanyl (C₇),bicyclo[2.2.2]octanyl (C₈), and the like. Exemplary C₃₋₁₀ carbocyclylgroups include, without limitation, the aforementioned C₃₋₈ carbocyclylgroups as well as cyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀),cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl(C₁₀), spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examplesillustrate, in certain embodiments, the carbocyclyl group is eithermonocyclic (“monocyclic carbocyclyl”) or contain a fused, bridged orspiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) andcan be saturated or can be partially unsaturated. “Carbocyclyl” alsoincludes ring systems wherein the carbocyclyl ring, as defined above, isfused with one or more aryl or heteroaryl groups wherein the point ofattachment is on the carbocyclyl ring, and in such instances, the numberof carbons continue to designate the number of carbons in thecarbocyclic ring system. In certain embodiments, each instance of acarbocyclyl group is independently optionally substituted, e.g.,unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents. In certainembodiments, the carbocyclyl group is unsubstituted C₃₋₁₀ carbocyclyl.In certain embodiments, the carbocyclyl group is a substituted C₃₋₁₀carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 10 ring carbon atoms (“C₃₋₁₀cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ringcarbon atoms (“C₃₋₈ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 6 ring carbon atoms (“C₃₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C₅₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ cycloalkyl”). Examples of C₅₋₆ cycloalkyl groupsinclude cyclopentyl (C₅) and cyclohexyl (C₅). Examples of C₃₋₆cycloalkyl groups include the aforementioned C₅₋₆ cycloalkyl groups aswell as cyclopropyl (C₃) and cyclobutyl (C₄). Examples of C₃₋₈cycloalkyl groups include the aforementioned C₃₋₆ cycloalkyl groups aswell as cycloheptyl (C₇) and cyclooctyl (C₈). In certain embodiments,each instance of a cycloalkyl group is independently unsubstituted (an“unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”)with one or more substituents. In certain embodiments, the cycloalkylgroup is unsubstituted C₃₋₁₀ cycloalkyl. In certain embodiments, thecycloalkyl group is substituted C₃₋₁₀ cycloalkyl.

“Heterocyclyl” or “heterocyclic” refers to a radical of a 3- to10-membered non-aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“3-10 membered heterocyclyl”). Inheterocyclyl groups that contain one or more nitrogen atoms, the pointof attachment can be a carbon or nitrogen atom, as valency permits. Aheterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”)or a fused, bridged or spiro ring system such as a bicyclic system(“bicyclic heterocyclyl”), and can be saturated or can be partiallyunsaturated. Heterocyclyl bicyclic ring systems can include one or moreheteroatoms in one or both rings. “Heterocyclyl” also includes ringsystems wherein the heterocyclyl ring, as defined above, is fused withone or more carbocyclyl groups wherein the point of attachment is eitheron the carbocyclyl or heterocyclyl ring, or ring systems wherein theheterocyclyl ring, as defined above, is fused with one or more aryl orheteroaryl groups, wherein the point of attachment is on theheterocyclyl ring, and in such instances, the number of ring memberscontinue to designate the number of ring members in the heterocyclylring system. In certain embodiments, each instance of heterocyclyl isindependently optionally substituted, e.g., unsubstituted (an“unsubstituted heterocyclyl”) or substituted (a “substitutedheterocyclyl”) with one or more substituents. In certain embodiments,the heterocyclyl group is unsubstituted 3-10 membered heterocyclyl. Incertain embodiments, the heterocyclyl group is substituted 3-10 memberedheterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 memberednon-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In someembodiments, a heterocyclyl group is a 5-8 membered non-aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl groupis a 5-6 membered non-aromatic ring system having ring carbon atoms and1-4 ring heteroatoms, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In someembodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, the 5-6 membered heterocyclyl has one ring heteroatomselected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing one heteroatominclude, without limitation, azirdinyl, oxiranyl, and thiorenyl.Exemplary 4-membered heterocyclyl groups containing one heteroatominclude, without limitation, azetidinyl, oxetanyl, and thietanyl.Exemplary 5-membered heterocyclyl groups containing one heteroatominclude, without limitation, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl,and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groupscontaining two heteroatoms include, without limitation, dioxolanyl,oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-memberedheterocyclyl groups containing three heteroatoms include, withoutlimitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary6-membered heterocyclyl groups containing one heteroatom include,without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl,and thianyl. Exemplary 6-membered heterocyclyl groups containing twoheteroatoms include, without limitation, piperazinyl, morpholinyl,dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groupscontaining two heteroatoms include, without limitation, triazinanyl.Exemplary 7-membered heterocyclyl groups containing one heteroatominclude, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary8-membered heterocyclyl groups containing one heteroatom include,without limitation, azocanyl, oxecanyl, and thiocanyl. Exemplary5-membered heterocyclyl groups fused to a C₆ aryl ring (also referred toherein as a 5,6-bicyclic heterocyclic ring) include, without limitation,indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl,benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groupsfused to an aryl ring (also referred to herein as a 6,6-bicyclicheterocyclic ring) include, without limitation, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and the like.

“Aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclicor tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14πelectrons shared in a cyclic array) having 6-14 ring carbon atoms andzero heteroatoms provided in the aromatic ring system (“C₆₋₁₄ aryl”). Insome embodiments, an aryl group has six ring carbon atoms (“C₆ aryl”;e.g., phenyl). In some embodiments, an aryl group has ten ring carbonatoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). Insome embodiments, an aryl group has fourteen ring carbon atoms (“C₁₋₄aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein thearyl ring, as defined above, is fused with one or more carbocyclyl orheterocyclyl groups wherein the radical or point of attachment is on thearyl ring, and in such instances, the number of carbon atoms continue todesignate the number of carbon atoms in the aryl ring system. In certainembodiments, each instance of an aryl group is independently optionallysubstituted, e.g., unsubstituted (an “unsubstituted aryl”) orsubstituted (a “substituted aryl”) with one or more substituents. Incertain embodiments, the aryl group is unsubstituted C₆₋₁₄ aryl. Incertain embodiments, the aryl group is substituted C₆₋₁₄ aryl.

“Heteroaryl” refers to a radical of a 5-10 membered monocyclic orbicyclic 4n+2 aromatic ring system (e.g., having 6 or 10π electronsshared in a cyclic array) having ring carbon atoms and 1-4 ringheteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from nitrogen, oxygen and sulfur(“5-10 membered heteroaryl”). In heteroaryl groups that contain one ormore nitrogen atoms, the point of attachment can be a carbon or nitrogenatom, as valency permits. Heteroaryl bicyclic ring systems can includeone or more heteroatoms in one or both rings. “Heteroaryl” includes ringsystems wherein the heteroaryl ring, as defined above, is fused with oneor more carbocyclyl or heterocyclyl groups wherein the point ofattachment is on the heteroaryl ring, and in such instances, the numberof ring members continue to designate the number of ring members in theheteroaryl ring system. “Heteroaryl” also includes ring systems whereinthe heteroaryl ring, as defined above, is fused with one or more arylgroups wherein the point of attachment is either on the aryl orheteroaryl ring, and in such instances, the number of ring membersdesignates the number of ring members in the fused (aryl/heteroaryl)ring system. Bicyclic heteroaryl groups wherein one ring does notcontain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and thelike) the point of attachment can be on either ring, e.g., either thering bearing a heteroatom (e.g., 2-indolyl) or the ring that does notcontain a heteroatom (e.g., 5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selectedfrom nitrogen, oxygen, and sulfur. In certain embodiments, each instanceof a heteroaryl group is independently optionally substituted, e.g.,unsubstituted (“unsubstituted heteroaryl”) or substituted (“substitutedheteroaryl”) with one or more substituents. In certain embodiments, theheteroaryl group is unsubstituted 5-14 membered heteroaryl. In certainembodiments, the heteroaryl group is substituted 5-14 memberedheteroaryl.

Exemplary 5-membered heteroaryl groups containing one heteroatominclude, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary5-membered heteroaryl groups containing two heteroatoms include, withoutlimitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryl groups containing threeheteroatoms include, without limitation, triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing fourheteroatoms include, without limitation, tetrazolyl. Exemplary6-membered heteroaryl groups containing one heteroatom include, withoutlimitation, pyridinyl. Exemplary 6-membered heteroaryl groups containingtwo heteroatoms include, without limitation, pyridazinyl, pyrimidinyl,and pyrazinyl. Exemplary 6-membered heteroaryl groups containing threeor four heteroatoms include, without limitation, triazinyl andtetrazinyl, respectively. Exemplary 7-membered heteroaryl groupscontaining one heteroatom include, without limitation, azepinyl,oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groupsinclude, without limitation, indolyl, isoindolyl, indazolyl,benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl,benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl,benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl,indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groupsinclude, without limitation, naphthyridinyl, pteridinyl, quinolinyl,isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

“Partially unsaturated” refers to a group that includes at least onedouble or triple bond. The term “partially unsaturated” is intended toencompass rings having multiple sites of unsaturation, but is notintended to include aromatic groups (e.g., aryl or heteroaryl groups) asherein defined. Likewise, “saturated” refers to a group that does notcontain a double or triple bond, i.e., contains all single bonds.

In some embodiments, aliphatic, alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, and heteroaryl groups, as defined herein, areoptionally substituted (e.g., “substituted” or “unsubstituted”aliphatic, “substituted” or “unsubstituted” alkyl, “substituted” or“unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl,“substituted” or “unsubstituted” carbocyclyl, “substituted” or“unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or“substituted” or “unsubstituted” heteroaryl group). In general, the term“substituted”, whether preceded by the term “optionally” or not, meansthat at least one hydrogen present on a group (e.g., a carbon ornitrogen atom) is replaced with a permissible substituent, e.g., asubstituent which upon substitution results in a stable compound, e.g.,a compound which does not spontaneously undergo transformation such asby rearrangement, cyclization, elimination, or other reaction. Unlessotherwise indicated, a “substituted” group has a substituent at one ormore substitutable positions of the group, and when more than oneposition in any given structure is substituted, the substituent iseither the same or different at each position. The term “substituted” iscontemplated to include substitution with all permissible substituentsof organic compounds, including any of the substituents described hereinthat results in the formation of a stable compound. The presentdisclosure contemplates any and all such combinations in order to arriveat a stable compound. For purposes of this disclosure, heteroatoms suchas nitrogen may have hydrogen substituents and/or any suitablesubstituent as described herein which satisfy the valencies of theheteroatoms and results in the formation of a stable moiety.

Exemplary carbon atom substituents include, but are not limited to,halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(aa), —ON(R^(bb))₂,—N(R^(bb))₂, —N(R^(bb))₃ ⁺X⁻, —N(OR^(cc))R^(bb), —SH, —SR^(aa),—SSR^(cc), —C(═O)R^(aa), —CO₂H, —CHO, —C(OR^(cc))₂, —CO₂R^(aa),—OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂, —OC(═O)N(R^(bb))₂,—NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —OC(═NR^(bb))N(R^(bb))₂,—NR^(bb)C(═NR^(bb))N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa),—NR^(bb)SO₂R^(aa), —SO₂N(R^(bb))₂, —SO₂R^(aa), —SO₂OR^(aa), —OSO₂R^(aa),—S(═O)R^(aa), —OS(═O)R^(aa), —Si(R^(aa))₃,—OSi(R^(aa))₃—C(═S)N(R^(bb))₂, —C(═O)SR^(aa), —C(═S)SR^(aa),—SC(═S)SR^(aa), —SC(═O)SR^(aa), —OC(═O)SR^(aa), —SC(═O)OR^(aa),—SC(═O)R^(aa), —P(═O)₂R^(aa), —OP(═O)₂R^(aa), —P(═O)(R^(aa))₂,—OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂,—OP(═O)₂N(R^(bb))₂, —P(═O)(NR^(bb))₂, —OP(═O)(NR^(bb))₂,—NR^(bb)P(═O)(OR^(cc))₂, —NR^(bb)P(═O)(NR^(bb))₂, —P(R^(cc))₂,—P(R^(cc))₃, —OP(R^(cc))₂, —OP(R^(cc))₃, —B(R^(aa))₂, —B(OR^(cc))₂,—BR^(aa)(OR^(cc)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

or two geminal hydrogens on a carbon atom are replaced with the group═O, ═S, ═NN(R^(bb))₂, ═NNR^(bb)C(═O)R^(aa), ═NNR^(bb)C(═O)OR^(aa),═NNR^(bb)S(═O)₂R^(aa), ═NR^(bb), or ═NOR^(cc);

each instance of R^(aa) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl,3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, ortwo R^(aa) groups are joined to form a 3-14 membered heterocyclyl or5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(bb) is, independently, selected from hydrogen, —OH,—OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂,—SO₂R^(cc), —SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc),—C(═S)SR^(cc), —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂,—P(═O)(NR^(cc))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and5-14 membered heteroaryl, or two R^(bb) groups are joined to form a 3-14membered heterocyclyl or 5-14 membered heteroaryl ring, wherein eachalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylis independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(cc) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl,alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(dd) is, independently, selected from halogen, —CN,—NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(ee), —ON(R^(ff))₂, —N(R^(ff))₂,—N(R^(ff))₃ ⁺X⁻, —N(OR^(ee))R^(ff), —SH, —SR^(ee), —SSR^(ee),—C(═O)R^(ee), —CO₂H, —CO₂R^(ee), —OC(═O)R^(ee), —OCO₂R^(ee),—C(═O)N(R^(ff))₂, —OC(═O)N(R^(ff))₂, —NR^(ff)C(═O)R^(ee),—NR^(ff)CO₂R^(ee), —NR^(ff)C(═O)N(R^(ff))₂, —C(═NR^(ff))OR^(ee),—OC(═NR^(ff))R^(ee), —OC(═NR^(ff))OR^(ee), —C(═NR^(ff))N(R^(ff))₂,—OC(═NR^(ff))N(R^(ff))₂, —NR^(ff)C(═NR^(ff))N(R^(ff))₂,NR^(ff)SO₂R^(ee), —SO₂N(R^(ff))₂, —SO₂R^(ee), —SO₂OR^(ee), —OSO₂R^(ee),—S(═O)R^(ee), —Si(R^(ee))₃, —OSi(R^(ee))₃, —C(═S)N(R^(ff))₂,—C(═O)SR^(ee), —C(═S)SR^(ee), —SC(═S)SR^(ee), —P(═O)₂R^(ee),—P(═O)(R^(ee))₂, —OP(═O)(R^(ee))₂, —OP(═O)(OR^(ee))₂, C₁₋₆ alkyl, C₁₋₆perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, 3-10membered heterocyclyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl, whereineach alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg)groups, or two geminal R^(dd) substituents can be joined to form ═O or═S;

each instance of R^(ee) is, independently, selected from C₁₋₆ alkyl,C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, C₆₋₁₀aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, whereineach alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg)groups;

each instance of R^(ff) is, independently, selected from hydrogen, C₁₋₆alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl,3-10 membered heterocyclyl, C₆₋₁₀ aryl and 5-10 membered heteroaryl, ortwo R^(ff) groups are joined to form a 3-14 membered heterocyclyl or5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups; and

each instance of R^(gg) is, independently, halogen, —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OC₁₋₆ alkyl, —ON(C₁₋₆ alkyl)₂, —N(C₁₋₆ alkyl)₂,—N(C₁₋₆ alkyl)₃ ⁺X⁻, —NH(C₁₋₅ alkyl)₂ ⁺X⁻, —NH₂(C₁₋₆ alkyl)⁺X⁻, —NH₃⁺X⁻, —N(OC₁₋₆ alkyl)(C₁₋₆ alkyl), —N(OH)(C₁₋₆ alkyl), —NH(OH), —SH,—SC₁₋₆ alkyl, —SS(C₁₋₆ alkyl), —C(═O)(C₁₋₆ alkyl), —CO₂H, —CO₂(C₁₋₆alkyl), —OC(═O)(C₁₋₆ alkyl), —OCO₂(C₁₋₆ alkyl), —C(═O)NH₂, —C(═O)N(C₁₋₆alkyl)₂, —OC(═O)NH(C₁₋₆ alkyl), —NHC(═O)(C₁₋₆ alkyl), —N(C₁₋₆alkyl)C(═O)(C₁₋₆ alkyl), —NHCO₂(C₁₋₅ alkyl), —NHC(═O)N(C₁₋₆ alkyl)₂,—NHC(═O)NH(C₁₋₆ alkyl), —NHC(═O)NH₂, —C(═NH)O(C₁₋₆ alkyl), —OC(═NH)(C₁₋₆alkyl), —OC(═NH)OC₁₋₆ alkyl, —C(═NH)N(C₁₋₆ alkyl)₂, —C(═NH)NH(C₁₋₆alkyl), —C(═NH)NH₂, —OC(═NH)N(C₁₋₆ alkyl)₂, —OC(NH)NH(C₁₋₆ alkyl),—OC(NH)NH₂, —NHC(NH)N(C₁₋₆ alkyl)₂, —NHC(═NH)NH₂, —NHSO₂(C₁₋₆ alkyl),—SO₂N(C₁₋₆ alkyl)₂, —SO₂NH(C₁₋₆ alkyl), —SO₂NH₂, —SO₂C₁₋₆ alkyl,—SO₂OC₁₋₆ alkyl, —OSO₂C₁₋₆ alkyl, —SOC₁₋₆ alkyl, —Si(C₁₋₆ alkyl)₃,—OSi(C₁₋₆ alkyl)₃—C(═S)N(C₁₋₆ alkyl)₂, C(═S)NH(C₁₋₆ alkyl), C(═S)NH₂,—C(═O)S(C₁₋₆ alkyl), —C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl, —P(═O)₂(C₁₋₆alkyl), —P(═O)(C₁₋₆ alkyl)₂, —OP(═O)(C₁₋₆ alkyl)₂, —OP(═O)(OC₁₋₆alkyl)₂, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,C₃₋₁₀ carbocyclyl, C₆₋₁₀ aryl, 3-10 membered heterocyclyl, 5-10 memberedheteroaryl; or two geminal R^(gg) substituents can be joined to form ═Oor ═S; wherein X⁻ is a counterion.

A “counterion” or “anionic counterion” is a negatively charged groupassociated with a cationic quaternary amino group in order to maintainelectronic neutrality. Exemplary counterions include halide ions (e.g.,F⁻, Cl⁻, Br⁻, I⁻), NO₃ ⁻, ClO₄ ⁻, OH⁻, H₂PO₄ ⁻, HSO₄ ⁻, sulfonate ions(e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate,benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate,ethanoate, propanoate, benzoate, glycerate, lactate, tartrate,glycolate, and the like).

“Halo” or “halogen” refers to fluorine (fluoro, —F), chlorine (chloro,—Cl), bromine (bromo, —Br), or iodine (iodo, —I).

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quarternary nitrogenatoms. Exemplary nitrogen atom substitutents include, but are notlimited to, hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),—C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(bb))R^(aa),—C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc),—SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc),—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂, —P(═O)(NR^(cc))₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups attached to a nitrogen atom are joinedto form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl,and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5R^(dd) groups, and wherein R^(aa), R^(bb), R^(cc) and R^(dd) are asdefined above.

In certain embodiments, the substituent present on a nitrogen atom is anitrogen protecting group (also referred to as an amino protectinggroup). Nitrogen protecting groups include, but are not limited to, —OH,—OR^(aa), —N(R^(cc))₂, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))R^(aa), —C(═NR^(cc))OR^(aa),—C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),—SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), C₁₋₁₀ alkyl(e.g., aralkyl, heteroaralkyl), C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aralkyl, aryl, and heteroaryl is independently substitutedwith 0, 1, 2, 3, 4, or 5 R^(dd) groups, and wherein R^(aa), R^(bb),R^(cc), and R^(dd) are as defined herein. Nitrogen protecting groups arewell known in the art and include those described in detail inProtecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts,3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

Amide nitrogen protecting groups (e.g., —C(═O)R^(aa)) include, but arenot limited to, formamide, acetamide, chloroacetamide,trichloroacetamide, trifluoroacetamide, phenylacetamide,3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide,N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide,o-nitophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide,(N′-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide,3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine,o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

Carbamate nitrogen protecting groups (e.g., —C(═O)OR^(aa)) include, butare not limited to, methyl carbamate, ethyl carbamante,9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethylcarbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzylcarbamate.

Sulfonamide nitrogen protecting groups (e.g., —S(═O)₂R^(aa)) include,but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to,phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacylderivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanylderivative, N-acetylmethionine derivative,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N-(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentaacylchromium-or tungsten)acyl]amine, N-copper chelate,N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, the substituent present on an oxygen atom is anoxygen protecting group (also referred to as a hydroxyl protectinggroup). Oxygen protecting groups include, but are not limited to,—R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa),—C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Oxygen protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

Exemplary oxygen protecting groups include, but are not limited to,methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodisulfuran-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts).

In certain embodiments, the substituent present on a sulfur atom is asulfur protecting group (also referred to as a thiol protecting group).Sulfur protecting groups include, but are not limited to, —R^(aa),—N(R^(bb))₂, —C(═O)SR^(aa), —CO₂R^(aa), —C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂,—S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃, —P(R^(cc))₂, —P(R^(cc))₃,—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and—P(═O)(NR^(bb))₂, wherein R^(aa), R^(bb), and R^(cc) are as definedherein. Sulfur protecting groups are well known in the art and includethose described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999,incorporated herein by reference.

These and other exemplary substituents are described in more detail inthe Detailed Description, Examples, and claims. The present disclosureis not intended to be limited in any manner by the above exemplarylisting of substituents.

“Pharmaceutically acceptable salt” refers to those salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and other animals without undue toxicity,irritation, allergic response, and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, Berge et al. describepharmaceutically acceptable salts in detail in J. PharmaceuticalSciences (1977) 66:1-19. Pharmaceutically acceptable salts of thecompounds describe herein include those derived from suitable inorganicand organic acids and bases. Examples of pharmaceutically acceptable,nontoxic acid addition salts are salts of an amino group formed withinorganic acids such as hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid and perchloric acid or with organic acids such asacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,succinic acid, or malonic acid or by using other methods used in the artsuch as ion exchange. Other pharmaceutically acceptable salts includeadipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, quaternary salts.

A “subject” to which administration is contemplated includes, but is notlimited to, humans (e.g., a male or female of any age group, e.g., apediatric subject (e.g, infant, child, adolescent) or adult subject(e.g., young adult, middle-aged adult or senior adult)) and/or othernon-human animals, for example, non-human mammals (e.g., primates (e.g.,cynomolgus monkeys, rhesus monkeys); commercially relevant mammals suchas cattle, pigs, horses, sheep, goats, cats, and/or dogs), birds (e.g.,commercially relevant birds such as chickens, ducks, geese, and/orturkeys), rodents (e.g., rats and/or mice), reptiles, amphibians, andfish. In certain embodiments, the non-human animal is a mammal. Thenon-human animal may be a male or female at any stage of development. Anon-human animal may be a transgenic animal.

“Condition,” “disease,” and “disorder” are used interchangeably herein.

“Treat,” “treating” and “treatment” encompasses an action that occurswhile a subject is suffering from a condition which reduces the severityof the condition or retards or slows the progression of the condition(“therapeutic treatment”). “Treat,” “treating” and “treatment” alsoencompasses an action that occurs before a subject begins to suffer fromthe condition and which inhibits or reduces the severity of thecondition (“prophylactic treatment”).

An “effective amount” of a compound refers to an amount sufficient toelicit the desired biological response, e.g., treat the condition. Aswill be appreciated by those of ordinary skill in this art, theeffective amount of a compound described herein may vary depending onsuch factors as the desired biological endpoint, the pharmacokinetics ofthe compound, the condition being treated, the mode of administration,and the age and health of the subject. An effective amount encompassestherapeutic and prophylactic treatment.

A “therapeutically effective amount” of a compound is an amountsufficient to provide a therapeutic benefit in the treatment of acondition or to delay or minimize one or more symptoms associated withthe condition. A therapeutically effective amount of a compound means anamount of therapeutic agent, alone or in combination with othertherapies, which provides a therapeutic benefit in the treatment of thecondition. The term “therapeutically effective amount” can encompass anamount that improves overall therapy, reduces or avoids symptoms orcauses of the condition, or enhances the therapeutic efficacy of anothertherapeutic agent.

A “prophylactically effective amount” of a compound is an amountsufficient to prevent a condition, or one or more symptoms associatedwith the condition or prevent its recurrence. A prophylacticallyeffective amount of a compound means an amount of a therapeutic agent,alone or in combination with other agents, which provides a prophylacticbenefit in the prevention of the condition. The term “prophylacticallyeffective amount” can encompass an amount that improves overallprophylaxis or enhances the prophylactic efficacy of anotherprophylactic agent.

As used herein, the term “methyltransferase” represents transferaseclass enzymes that are able to transfer a methyl group from a donormolecule to an acceptor molecule, e.g., an amino acid residue of aprotein or a nucleic base of a DNA molecule. Methylransferases typicallyuse a reactive methyl group bound to sulfur in S-adenosyl methionine(SAM) as the methyl donor. In some embodiments, a methyltransferasedescribed herein is a protein methyltransferase. In some embodiments, amethyltransferase described herein is a histone methyltransferase.Histone methyltransferases (HMT) are histone-modifying enzymes,(including histone-lysine N-methyltransferase and histone-arginineN-methyltransferase), that catalyze the transfer of one or more methylgroups to lysine and arginine residues of histone proteins. In certainembodiments, a methyltransferase described herein is a histone-arginineN-methyltransferase.

As generally described above, provided herein are compounds useful asPRMT5 inhibitors. In some embodiments, the present disclosure provides acompound of Formula (I):

or a pharmaceutically acceptable salt thereof,wherein

represents a single or double bond;

R¹ is hydrogen, R^(z), or —C(O)R^(z), wherein R^(z) is optionallysubstituted C₁₋₆ alkyl;

L is —N(R)C(O)—, —C(O)N(R)—, —N(R)C(O)N(R)—,—N(R)C(O)O—, or —OC(O)N(R)—;

each R is independently hydrogen or optionally substituted C₁₋₆aliphatic;

Ar is a monocyclic or bicyclic aromatic ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, and sulfur, wherein Ar issubstituted with 0, 1, 2, 3, 4, or 5 R^(y) groups, as valency permits;

each R^(y) is independently selected from the group consisting of halo,—CN, —NO₂, optionally substituted aliphatic, optionally substitutedcarbocyclyl, optionally substituted aryl, optionally substitutedheterocyclyl, optionally substituted heteroaryl, —OR^(A), —N(R^(B))₂,—SR^(A), —C(═O)R^(A), —C(O)OR^(A), —C(O)SR^(A), —C(O)N(R^(B))₂,—C(O)N(R^(B))N(R^(B))₂, —OC(O)R^(A), —OC(O)N(R^(B))₂, —NR^(B)C(O)R^(A),—NR^(B)C(O)N(R^(B))₂, —NR^(B)C(O)N(R^(B))N(R^(B))₂, —NR^(B)C(O)OR^(A),—SC(O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A),—C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B), —C(═S)R^(A),—C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(O)R^(A), —OS(O)₂R^(A), —SO₂R^(A),—NR^(B)SO₂R^(A), or —SO₂N(R^(B))₂;

each R^(A) is independently selected from the group consisting ofhydrogen, optionally substituted aliphatic, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, and optionally substituted heteroaryl;

each R^(B) is independently selected from the group consisting ofhydrogen, optionally substituted aliphatic, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, and optionally substituted heteroaryl, or two R^(B) groups aretaken together with their intervening atoms to form an optionallysubstituted heterocyclic ring;

R⁵, R⁶, R⁷, and R⁸ are independently hydrogen, halo, or optionallysubstituted aliphatic;

each R^(x) is independently selected from the group consisting of halo,—CN, optionally substituted aliphatic, —OR′, and —N(R″)₂;

R′ is hydrogen or optionally substituted aliphatic;

each R″ is independently hydrogen or optionally substituted aliphatic,or two R″ are taken together with their intervening atoms to form aheterocyclic ring; and

n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, as valency permits.

In certain embodiments, a provided compound is of Formula (I-a):

or a pharmaceutically acceptable salt thereof, wherein R¹, R⁵, R⁶, R⁷,R⁸, R^(x), n, L, and Ar are as described herein.

In certain embodiments, a provided compound is of Formula (I-b):

or a pharmaceutically acceptable salt thereof, wherein R¹, R⁵, R⁶, R⁷,R⁸, R^(x), n, L, and Ar are as described herein.

In certain embodiments, a provided compound is of Formula (I-c):

or a pharmaceutically acceptable salt thereof, wherein R¹, R^(x), n, L,and Ar are as described herein.

In certain embodiments, a provided compound is of Formula (I′):

or a pharmaceutically acceptable salt thereof, wherein R¹, R^(x), n, L,and Ar are as described herein.

In certain embodiments, a provided compound is of Formula (I′-a):

or a pharmaceutically acceptable salt thereof, wherein R¹, R^(x), n, L,and Ar are as described herein.

In certain embodiments, a provided compound is of Formula (I′-b):

or a pharmaceutically acceptable salt thereof, wherein R¹, R^(x), n, L,and Ar are as described herein.

In certain embodiments, a provided compound is of Formula (II):

or a pharmaceutically acceptable salt thereof, wherein R¹, R^(x), n, andAr are as described herein.

In certain embodiments, a provided compound is of Formula (II-a):

or a pharmaceutically acceptable salt thereof, wherein R¹, R^(x), n, andAr are as described herein.

In certain embodiments, a provided compound is of Formula (II-b):

or a pharmaceutically acceptable salt thereof, wherein R¹, R^(x), n, andAr are as described herein.

In certain embodiments, a provided compound is of Formula (III):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (III-a):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (III-b):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (IV):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (IV-a):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (IV-b):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (V):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (V-a):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (V-b):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (VI):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (VI-a):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (VI-b):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (VII):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (VII-a):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (VII-b):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (VIII):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (VIII-a):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (VIII-b):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (IX):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (IX-a):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (IX-b):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (X):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (X-a):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (X-b):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (XI):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (XI-a):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (XI-b):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (XII):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (XII-a):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (XII-b):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (XIII):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (XIII-a):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

In certain embodiments, a provided compound is of Formula (XIII-b):

or a pharmaceutically acceptable salt thereof, wherein R^(y) is asdescribed herein.

As defined generally above, R¹ is hydrogen, R^(z), or —C(O)R^(z),wherein R^(z) is optionally substituted C₁₋₆ alkyl. In certainembodiments, R¹ is hydrogen. In some embodiments, R¹ is optionallysubstituted C₁₋₆ alkyl. In certain embodiments, R¹ is unsubstituted C₁₋₆alkyl. In certain embodiments, R¹ is methyl, ethyl, or propyl. In someembodiments, R¹ is —C(O)R^(z), wherein R^(z) is optionally substitutedC₁₋₆ alkyl. In certain embodiments, R¹ is —C(O)R^(z), wherein R^(z) isunsubstituted C₁₋₆ alkyl. In certain embodiments, R¹ is acetyl.

As defined generally above, L is —N(R)C(O)—, —C(O)N(R)—, —N(R)C(O)N(R)—,—N(R)C(O)O—, or —OC(O)N(R)—, wherein R is as described herein. In someembodiments, L is —N(R)C(O)—. In some embodiments, L is —NHC(O)—. Insome embodiments, L is —N(C₁₋₆ alkyl)C(O)—. In some embodiments, L is—N(CH₃)C(O)—. In some embodiments, L is —C(O)N(R)—. In some embodiments,L is —C(O)NH—. In some embodiments, L is —C(O)N(C₁₋₆ alkyl)-. In someembodiments, L is —C(O)N(CH₃)—. In some embodiments, L is—N(R)C(O)N(R)—. In some embodiments, L is —NHC(O)NH—. In someembodiments, L is —NHC(O)N(R)—. In some embodiments, L is —N(R)C(O)NH—.In some embodiments, L is —N(CH₃)C(O)N(R)—. In some embodiments, L is—N(R)C(O)N(CH₃)—. In some embodiments, L is —N(CH₃)C(O)N(CH₃)—. In someembodiments, L is —N(R)C(O)O—. In some embodiments, L is —NHC(O)O—. Insome embodiments, L is —N(C₁₋₆ alkyl)C(O)O—. In some embodiments, L is—N(CH₃)C(O)O—. In some embodiments, L is —OC(O)N(R)—. In someembodiments, L is —OC(O)NH—. In some embodiments, L is —OC(O)N(C₁₋₆alkyl)-. In some embodiments, L is —OC(O)N(CH₃)—.

As defined generally above, each R is independently hydrogen oroptionally substituted C₁₋₆ aliphatic. In certain embodiments, R ishydrogen. In some embodiments, R is optionally substituted C₁₋₆aliphatic. In some embodiments, R is substituted C₁₋₆ aliphatic. In someembodiments, R is unsubstituted C₁₋₆ aliphatic. In some embodiments, Ris optionally substituted C₁₋₆ alkyl. In some embodiments, R issubstituted C₁₋₆ alkyl. In some embodiments, R is unsubstituted C₁₋₆alkyl. In some embodiments, R is methyl, ethyl, or propyl.

As defined generally above, Ar is a monocyclic or bicyclic aromatic ringhaving 0-4 heteroatoms independently selected from nitrogen, oxygen, andsulfur, wherein Ar is substituted with 0, 1, 2, 3, 4, or 5 R^(y) groups,as valency permits. For avoidance of confusion, though Ar is sometimesused to denote the element argon, as used herein it denotes a monocyclicor bicyclic aromatic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur, wherein Ar is substituted with 0, 1,2, 3, 4, or 5 R^(y) groups, as valency permits, and various embodimentsthereof as described herein. In certain embodiments, Ar isunsubstituted. In certain embodiments, Ar is substituted with one or twoR^(y) groups. In certain embodiments, Ar is substituted with one R^(y)group. In certain embodiments, Ar is substituted with two R^(y) groups.In certain embodiments, Ar is substituted with three R^(y) groups. Incertain embodiments, Ar is substituted with four R^(y) groups. Incertain embodiments, Ar is substituted with five R^(y) groups.

In certain embodiments, Ar is phenyl substituted with 0, 1, 2, 3, 4, or5 R^(y) groups. In certain embodiments, Ar is phenyl substituted withone or two R^(y) groups. In certain embodiments, Ar is unsubstitutedphenyl. In certain embodiments, Ar is phenyl substituted with one R^(y)group. In certain embodiments, Ar is phenyl substituted with two R^(y)groups. In certain embodiments, Ar is phenyl substituted with threeR^(y) groups. In certain embodiments, Ar is phenyl substituted with fourR^(y) groups. In certain embodiments, Ar is phenyl substituted with fiveR^(y) groups.

In certain embodiments, Ar is heteroaryl substituted with 0, 1, 2, 3, 4,or 5 R^(y) groups, as valency permits. In certain embodiments, Ar is a5- to 6-membered heteroaryl having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur, and is substituted with 0,1, 2, 3, or 4 R^(y) groups. In certain embodiments, Ar is anunsubstituted 5- to 6-membered heteroaryl having 1-3 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In certainembodiments, Ar is a 5- to 6-membered heteroaryl having 1-3 heteroatomsindependently selected from nitrogen, oxygen, and sulfur, and issubstituted with one or two R^(y) groups. In certain embodiments, Ar isa 5- to 6-membered heteroaryl having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur, and is substituted with oneR^(y) group. In certain embodiments, Ar is a 5-membered heteroarylhaving 1-3 heteroatoms independently selected from nitrogen, oxygen, andsulfur (e.g., furanyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl,thiazolyl, imidazolyl, pyrazolyl, isothiazolyl, triazolyl, oxadiazolyl,thiadiazolyl), and is substituted with 0, 1, 2, or 3 R^(y) groups. Incertain embodiments, Ar is a 6-membered heteroaryl having 1-3 nitrogens(e.g., pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazinyl), and issubstituted with 0, 1, 2, 3, or 4 R^(y) groups. In certain embodiments,Ar is pyridyl, and is substituted with 0, 1, 2, 3, or 4 R^(y) groups. Incertain embodiments, Ar is pyridyl, and is substituted with one R^(y)group. In certain embodiments, Ar is pyridyl, and is substituted withtwo R^(y) groups. In certain embodiments, Ar is a 6-membered heteroarylhaving two nitrogens (e.g., pyrimidyl, pyridazinyl, pyrazinyl), and issubstituted with 0, 1, 2, or 3 R^(y) groups. In certain embodiments, Aris a 6-membered heteroaryl having two nitrogens (e.g., pyrimidyl,pyridazinyl, pyrazinyl), and is substituted with one R^(y) group. Incertain embodiments, Ar is a 6-membered heteroaryl having two nitrogens(e.g., pyrimidyl, pyridazinyl, pyrazinyl), and is substituted with twoR^(y) groups.

In certain embodiments, Ar is a bicyclic aromatic ring having 0-4heteroatoms independently selected from nitrogen, oxygen, and sulfur,wherein Ar is substituted with 0, 1, 2, 3, or 4 R^(y) groups. In certainembodiments, Ar is an 8- to 12-membered bicyclic aromatic ring having0-4 heteroatoms independently selected from nitrogen, oxygen, andsulfur, wherein Ar is substituted with 0, 1, 2, 3, or 4 R^(y) groups. Incertain embodiments, Ar is an unsubstituted bicyclic aromatic ringhaving 0-4 heteroatoms independently selected from nitrogen, oxygen, andsulfur. In certain embodiments, Ar is a bicyclic aromatic ring having0-4 heteroatoms independently selected from nitrogen, oxygen, andsulfur, wherein Ar is substituted with one or two R^(y) groups. Incertain embodiments, Ar is a bicyclic aromatic ring having 0-4heteroatoms independently selected from nitrogen, oxygen, and sulfur,wherein Ar is substituted with one R^(y) group. In certain embodiments,Ar is a bicyclic aromatic ring having 0-4 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur, wherein Ar is substitutedwith two R^(y) groups. In certain embodiments, Ar is a bicyclic aromaticring having 0-4 heteroatoms independently selected from nitrogen,oxygen, and sulfur, wherein Ar is substituted with three R^(y) groups.In certain embodiments, Ar is a bicyclic aromatic ring having 0-4heteroatoms independently selected from nitrogen, oxygen, and sulfur,wherein Ar is substituted with four R^(y) groups. In certainembodiments, Ar is a bicyclic aromatic ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, and sulfur, wherein Ar issubstituted with five R^(y) groups. In certain embodiments, Ar isnaphthalene substituted with 0, 1, 2, 3, 4, or 5 R^(y) groups.

In certain embodiments, Ar is an 8- to 10-membered bicyclic heteroarylhaving 1-4 heteroatoms independently selected from nitrogen, oxygen, andsulfur, wherein Ar is substituted with 0, 1, 2, 3, or 4 R^(y) groups. Incertain embodiments, Ar is a 9-membered bicyclic heteroaryl having 1-3heteroatoms independently selected from nitrogen, oxygen, and sulfur(e.g., indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl,isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl,benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl,benzisothiazolyl, benzthiadiazolyl, indolizinyl), wherein Ar issubstituted with 0, 1, 2, 3, 4, or 5 R^(y) groups. In certainembodiments, Ar is a 10-membered bicyclic heteroaryl having 1-3heteroatoms independently selected from nitrogen, oxygen, and sulfur(e.g., naphthyridinyl, quinolinyl, isoquinolinyl, quinoxalinyl,quinazolinyl), wherein Ar is substituted with 0, 1, 2, 3, 4, or 5 R^(y)groups. In certain embodiments, Ar is selected from the group consistingof quinoline, benzimidazole, benzopyrazole, quinoxaline,tetrahydroquinoline, tetrahydroisoquinoline, naphthalene,tetrahydronaphthalene, 2,3-dihydrobenzo[b][1,4]dioxine, isoindole,2H-benzo[b][1,4]oxazin-3(4H)-one, 3,4-dihydro-2H-benzo[b][1,4]oxazine,and quinoxalin-2(1H)-one, wherein Ar is substituted with 0, 1, 2, 3, or4 R^(y) groups. In some embodiments, Ar is quinoline, wherein Ar issubstituted with 0, 1, 2, 3, or 4 R^(y) groups.

As defined generally above, each R^(y) is independently selected fromthe group consisting of halo, —CN, —NO₂, optionally substitutedaliphatic, optionally substituted carbocyclyl, optionally substitutedaryl, optionally substituted heterocyclyl, optionally substitutedheteroaryl, —OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A), —C(O)OR^(A),—C(O)SR^(A), —C(O)N(R^(B))₂, —C(O)N(R^(B))N(R^(B))₂, —OC(O)R^(A),—OC(O)N(R^(B))₂, —NR^(B)C(O)R^(A), —NR^(B)C(O)N(R^(B))₂,—NR^(B)C(O)N(R^(B))N(R^(B))₂, —NR^(B)C(O)OR^(A), —SC(O)R^(A),—C(═NR^(B))R^(A), —C(═NNR^(B))R^(A), —C(═NOR^(A))R^(A),—C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B), —C(═S)R^(A),—C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(O)R^(A), —OS(O)₂R^(A), —SO₂R^(A),—NR^(B)SO₂R^(A), or —SO₂N(R^(B))₂, wherein R^(A) and R^(B) are describedherein. In some embodiments, each R^(y) is independently selected fromthe group consisting of halo, —CN, —NO₂, optionally substitutedaliphatic, optionally substituted carbocyclyl, optionally substitutedphenyl, optionally substituted heterocyclyl, optionally substitutedheteroaryl, —OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A), —C(O)OR^(A),—C(O)SR^(A), —C(O)N(R^(B))₂, —OC(O)R^(A), —NR^(B)C(O)R^(A),—NR^(B)C(O)N(R^(B))₂, —SC(O)R^(A), —C(═NR^(B))R^(A),—C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B), —C(═S)R^(A),—C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(O)R^(A), —SO₂R^(A),—NR^(B)SO₂R^(A), and —SO₂N(R^(B))₂, wherein R^(A) and R^(B) aredescribed herein.

In some embodiments, at least one R^(y) is halo. In certain embodiments,at least one R^(y) is fluoro. In certain embodiments, at least one R^(y)is chloro. In some embodiments, at least one R^(y) is —CN. In someembodiments, at least one R^(y) is —OR^(A), wherein R^(A) is optionallysubstituted aliphatic. In some embodiments, at least one R^(y) is—OR^(A), wherein R^(A) is unsubstituted C₁₋₆ alkyl. In certainembodiments, at least one R^(y) is methoxy, ethoxy, or propoxy. Incertain embodiments, at least one R^(y) is methoxy. In some embodiments,at least one R^(y) is —OR^(A), wherein R^(A) is substituted C₁₋₆ alkyl.In certain embodiments, at least one R^(y) is —OCH₂CH₂N(CH₃)₂. In someembodiments, at least one R^(y) is —OR^(A), wherein R^(A) is optionallysubstituted heterocyclyl. In some embodiments, at least one R^(y) is—OR^(A), wherein R^(A) is an optionally substituted 4- to 7-memberedheterocyclyl having 1-2 heteroatoms independently selected fromnitrogen, oxygen, and sulfur. In some embodiments, at least one R^(y) is—OR^(A), wherein R^(A) is oxetanyl, tetrahydrofuranyl, ortetrahydropyranyl. In some embodiments, at least one R^(y) is—N(R^(B))₂. In some embodiments, at least one R^(y) is —N(R^(B))₂,wherein each R^(B) is independently selected from hydrogen or C₁₋₆alkyl. In some embodiments, at least one R^(y) is —NHR^(B). In someembodiments, at least one R^(y) is —N(C₁₋₆ alkyl)₂, —NH(C₁₋₆ alkyl), or—NH₂. In certain embodiments, at least one R^(y) is —NH₂. In certainembodiments, at least one R^(y) is —NHCH₃. In certain embodiments, atleast one R^(y) is —N(CH₃)₂. In some embodiments, at least one R^(y) is—N(R^(B))₂ or —NHR^(B), wherein at least one R^(B) is optionallysubstituted heterocyclyl. In some embodiments, at least one R^(y) is—N(R^(B))₂ or —NHR^(B), wherein at least one R^(B) is an optionallysubstituted 4- to 7-membered heterocyclyl having 1-2 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, at least one R^(y) is —N(R^(B))₂ or —NHR^(B), wherein atleast one R^(B) is oxetanyl, tetrahydropyranyl, or tetrahydrofuranyl. Insome embodiments, at least one R^(y) is —N(R^(B))₂ or —NHR^(B), whereinat least one R^(B) is optionally substituted piperidinyl or optionallysubstituted piperazinyl.

In some embodiments, at least one R^(y) is optionally substitutedaliphatic. In certain embodiments, at least one R^(y) is substitutedaliphatic. In certain embodiments, at least one R^(y) is unsubstitutedaliphatic. In some embodiments, at least one R^(y) is optionallysubstituted C₁₋₆ alkyl. In certain embodiments, at least one R^(y) isunsubstituted C₁₋₆ alkyl. In certain embodiments, at least one R^(y) issubstituted C₁₋₆ alkyl. In certain embodiments, at least one R^(y) ismethyl, ethyl, or propyl. In certain embodiments, at least one R^(y) ismethyl. In certain embodiments, at least one R^(y) is —CF₃, CHF₂, orCH₂F. In certain embodiments, at least one R^(y) is C₁₋₆ alkylsubstituted with aryl, heteroaryl, or heterocyclyl. In certainembodiments, at least one R^(y) is benzyl. In certain embodiments, atleast one R^(y) is —(C₁₋₆ alkyl)-aryl. In certain embodiments, at leastone R^(y) is —(C₁₋₆ alkyl)-heteroaryl. In certain embodiments, at leastone R^(y) is —(C₁₋₆ alkyl)-heterocyclyl. In certain embodiments, atleast one R^(y) is —CH₂-aryl. In certain embodiments, at least one R^(y)is —CH₂-heteroaryl. In certain embodiments, at least one R^(y) is—CH₂-heterocyclyl.

In some embodiments, at least one R^(y) is —C(O)N(R^(B))₂. In certainembodiments, at least one R^(y) is —C(O)NHR^(B). In certain embodiments,at least one R^(y) is —C(O)NH₂. In certain embodiments, at least oneR^(y) is —C(O)N(R^(B))₂, wherein the R^(B) groups are taken togetherwith their intervening atoms to form an optionally substituted 5- to6-membered heterocyclyl. In certain embodiments, at least one R^(y) is—C(O)N(R^(B))₂, wherein the R^(B) groups are taken together with theirintervening atoms to form an optionally substituted morpholinyl.

In some embodiments, at least one R^(y) is —SO₂N(R^(B))₂. In certainembodiments, at least one R^(y) is —SO₂NHR^(B). In certain embodiments,at least one R^(y) is —SO₂NH₂. In certain embodiments, at least oneR^(y) is —SO₂N(R^(B))₂, wherein neither R^(B) is hydrogen. In certainembodiments, at least one R^(y) is —SO₂NH(C₁₋₆ alkyl) or —SO₂N(C₁₋₆alkyl)₂. In certain embodiments, at least one R^(y) is —SO₂N(CH₃)₂. Incertain embodiments, at least one R^(y) is —SO₂N(R^(B))₂, wherein theR^(B) groups are taken together with their intervening atoms to form anoptionally substituted 5- to 6-membered heterocyclyl. In certainembodiments, at least one R^(y) is —SO₂-morpholinyl. In certainembodiments, at least one R^(y) is —SO₂-piperidinyl, —SO₂-piperazinyl,or —SO₂-piperidinyl.

In some embodiments, at least one R^(y) is —SO₂R^(A). In someembodiments, at least one

R^(y) is —SO₂R^(A), wherein R^(A) is optionally substituted aliphatic.In some embodiments, at least one R^(y) is —SO₂(C₁₋₆ alkyl). In someembodiments, at least one R^(y) is —SO₂CH₃. In some embodiments, atleast one R^(y) is —C(O)R^(A). In some embodiments, at least one R^(y)is —C(O)R^(A), wherein R^(A) is optionally substituted aliphatic. Insome embodiments, at least one R^(y) is —C(O)(C₁₋₆ alkyl). In someembodiments, at least one R^(y) is —C(O)CH₃.

In some embodiments, at least one R^(y) is —N(R^(B))C(O)R^(A). Incertain embodiments, at least one R^(y) is —NHC(O)R^(A). In certainembodiments, at least one R^(y) is —NHC(O)(C₁₋₆ alkyl). In certainembodiments, at least one R^(y) is —NHC(O)CH₃.

In some embodiments, at least one R^(y) is —N(R^(B))SO₂R^(A). In someembodiments, at least one R^(y) is —NHSO₂R^(A). In some embodiments, atleast one R^(y) is —N(C₁₋₆ alkyl)SO₂R^(A). In certain embodiments, atleast one R^(y) is —NHSO₂(C₁₋₆ alkyl) or —N(C₁₋₆ alkyl)SO₂(C₁₋₆ alkyl).In certain embodiments, at least one R^(y) is —NHSO₂CH₃. In certainembodiments, at least one R^(y) is —N(CH₃)SO₂CH₃.

In some embodiments, at least one R^(y) is optionally substitutedheterocyclyl, optionally substituted carbocyclyl, optionally substitutedaryl, or optionally substituted heteroaryl. In certain embodiments, atleast one R^(y) is an optionally substituted 5- to 6-memberedheterocyclyl having 1-2 heteroatoms independently selected fromnitrogen, oxygen, and sulfur. In certain embodiments, at least one R^(y)is an optionally substituted 5-membered heterocyclyl having oneheteroatom selected from nitrogen, oxygen, and sulfur. In certainembodiments, at least one R^(y) is optionally substituted pyrrolidinyl.In certain embodiments, at least one R^(y) is pyrroldinyl,hydroxypyrrolidinyl, or methylpyrrolidinyl. In certain embodiments, atleast one R^(y) is an optionally substituted 6-membered heterocyclylhaving 1-2 heteroatoms independently selected from nitrogen, oxygen, andsulfur. In certain embodiments, at least one R^(y) is an optionallysubstituted 6-membered heterocyclyl having one heteroatom selected fromnitrogen, oxygen, and sulfur. In certain embodiments, at least one R^(y)is optionally substituted piperidinyl. In certain embodiments, at leastone R^(y) is an optionally substituted 6-membered heterocyclyl havingtwo heteroatoms independently selected from nitrogen, oxygen, andsulfur. In certain embodiments, at least one R^(y) is optionallysubstituted piperidinyl, optionally substituted piperazinyl, oroptionally substituted morpholinyl. In certain embodiments, at least oneR^(y) is morpholinyl, tetrahydropyranyl, piperidinyl, methylpiperidinyl,piperazinyl, methylpiperazinyl, acetylpiperazinyl,methylsulfonylpiperazinyl, aziridinyl, or methylaziridinyl. In someembodiments, at least one R^(y) is an optionally substituted 5- to6-membered heteroaryl having 1-3 heteroatoms independently selected fromnitrogen, oxygen, and sulfur. In certain embodiments, at least one R^(y)is an optionally substituted 5-membered heteroaryl having 1-3heteroatoms independently selected from nitrogen, oxygen, and sulfur. Incertain embodiments, at least one R^(y) is an optionally substituted5-membered heteroaryl having one heteroatom selected from nitrogen,oxygen, and sulfur. In certain embodiments, at least one R^(y) is anoptionally substituted 5-membered heteroaryl having two heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In certainembodiments, at least one R^(y) is an optionally substituted 6-memberedheteroaryl having 1-3 nitrogens. In certain embodiments, at least oneR^(y) is an optionally substituted pyrazolyl. In certain embodiments, atleast one R^(y) is an optionally substituted imidazolyl. In certainembodiments, at least one R^(y) is an optionally substituted pyridyl. Incertain embodiments, at least one R^(y) is an optionally substitutedpyrimidyl. In certain embodiments, at least one R^(y) is pyrazolyl,methylpyrazolyl, imidazolyl, or methylimidazolyl.

In some embodiments, R^(y) is —OR^(A). In some embodiments, R^(y) is—OR^(A), wherein R^(A) is optionally substituted heterocyclyl. In someembodiments, R^(y) is —OR^(A), wherein R^(A) is optionally substitutedheteroaryl. In some embodiments, R^(y) is —OR^(A), wherein R^(A) isoptionally substituted cycloalkyl. In some embodiments, R^(y) is—N(R^(B))₂. In some embodiments, R^(y) is —NHR^(B). In some embodiments,R^(y) is —NHR^(B), wherein R^(B) is optionally substituted heterocyclyl.In some embodiments, R^(y) is —NHR^(B), wherein R^(B) is optionallysubstituted heteroaryl. In some embodiments, R^(y) is —NHR^(B), whereinR^(B) is optionally substituted cycloalkyl. In some embodiments, R^(y)is —N(R^(B))₂, wherein one R^(B) is optionally substituted heterocyclyl,and the other R^(B) is C₁₋₄ alkyl. In some embodiments, R^(y) is—N(R^(B))₂, wherein one R^(B) is optionally substituted heteroaryl, andthe other R^(B) is C₁₋₄ alkyl. In some embodiments, R^(y) is —N(R^(B))₂,wherein one R^(B) is optionally substituted cycloalkyl, and the otherR^(B) is C₁₋₄ alkyl.

In certain embodiments, Ar is selected from the group consisting of:

In certain embodiments, Ar is selected from the group consisting of:

As defined generally above, each R^(x) is independently selected fromthe group consisting of halo, —CN, optionally substituted aliphatic,—OR′, and —N(R″)₂. In certain embodiments, at least one R^(x) is halo.In certain embodiments, at least one R^(x) is fluoro. In certainembodiments, at least one R^(x) is —CN. In certain embodiments, at leastone R^(x) is optionally substituted aliphatic. In certain embodiments,at least one R^(x) is optionally substituted C₁₋₆ alkyl. In certainembodiments, at least one R^(x) is methyl. In certain embodiments, atleast one R^(x) is —CF₃. In certain embodiments, at least one R^(x) is—OR′ or —N(R″)₂. In certain embodiments, R^(x) is not —OR′ or —N(R″)₂.In certain embodiments, at least one R^(x) is —OCH₃. In certainembodiments, R^(x) is not —OCH₃.

One of ordinary skill in the art will appreciate that an R^(x) group canbe attached anywhere on the tetrahydroisoquinoline ordihydroisoquinoline ring. In certain embodiments, an R^(x) group isattached to the benzene portion of the tetrahydroisoquinoline ordihydroisoquinoline ring. In certain embodiments, an R^(x) group isattached to the tetrahydropyridine or dihydropyridine portion of thetetrahydroisoquinoline or dihydroisoquinoline ring. In certainembodiments, R^(x) groups are attached to both the benzene portion andthe tetrahydropyridine (or dihydropyridine) portion of thetetrahydroisoquinoline (or dihydroisoquinoline) ring. See, for example,the structures shown below:

In certain embodiments, a provided compound is of Formula (XIV):

or a pharmaceutically acceptable salt thereof.

As defined generally above, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Incertain embodiments, n is 0. In certain embodiments, n is 1. In certainembodiments, n is 2.

In certain embodiments, a provided compound is of Formula (XV), (XVI),(XVII), or (XVIII):

or a pharmaceutically acceptable salt thereof, wherein each R^(y) forFormula (XV), (XVI), (XVII), or (XVIII) is independently as describedherein.

In certain embodiments, a provided compound is of Formula (XV-a),(XVI-a), (XVII-a), or (XVIII-a):

or a pharmaceutically acceptable salt thereof, wherein R^(y) for Formula(XV-a), (XVI-a), (XVII-a), or (XVIII-a) is as described herein. In someembodiments, e.g. for Formula (XV-a), (XVI-a), (XVII-a), or (XVIII-a),R^(y) is —OR^(A). In some embodiments, e.g. for Formula (XV-a), (XVI-a),(XVII-a), or (XVIII-a), R^(y) is —OR^(A), wherein R^(A) is optionallysubstituted heterocyclyl. In some embodiments, e.g. for Formula (XV-a),(XVI-a), (XVII-a), or (XVIII-a), R^(y) is —OR^(A), wherein R^(A) isoptionally substituted heteroaryl. In some embodiments, e.g. for Formula(XV-a), (XVI-a), (XVII-a), or (XVIII-a), R^(y) is —OR^(A), wherein R^(A)is optionally substituted cycloalkyl. In some embodiments, e.g. forFormula (XV-a), (XVI-a), (XVII-a), or (XVIII-a), R^(y) is —N(R^(B))₂. Insome embodiments, e.g. for Formula (XV-a), (XVI-a), (XVII-a), or(XVIII-a), R^(y) is —NHR^(B). In some embodiments, e.g. for Formula(XV-a), (XVI-a), (XVII-a), or (XVIII-a), R^(y) is —NHR^(B), whereinR^(B) is optionally substituted heterocyclyl. In some embodiments, e.g.for Formula (XV-a), (XVI-a), (XVII-a), or (XVIII-a), R^(y) is —NHR^(B),wherein R^(B) is optionally substituted heteroaryl. In some embodiments,e.g. for Formula (XV-a), (XVI-a), (XVII-a), or (XVIII-a), R^(y) is—NHR^(B), wherein R^(B) is optionally substituted cycloalkyl. In someembodiments, e.g. for Formula (XV-a), (XVI-a), (XVII-a), or (XVIII-a),R^(y) is —N(R^(B))₂, wherein one R^(B) is optionally substitutedheterocyclyl, and the other R^(B) is C₁₋₄ alkyl. In some embodiments,e.g. for Formula (XV-a), (XVI-a), (XVII-a), or (XVIII-a), R^(y) is—N(R^(B))₂, wherein one R^(B) is optionally substituted heteroaryl, andthe other R^(B) is C₁₋₄ alkyl. In some embodiments, e.g. for Formula(XV-a), (XVI-a), (XVII-a), or (XVIII-a), R^(y) is —N(R^(B))₂, whereinone R^(B) is optionally substituted cycloalkyl, and the other R^(B) isC₁₋₄ alkyl.

In certain embodiments, a provided compound is of Formula (XV-b):

or a pharmaceutically acceptable salt thereof, wherein each R^(y) isindependently as described herein.

In some embodiments, e.g. for Formula (XV), (XVI), (XVII), (XVIII),(XV-a), (XVI-a), (XVII-a), (XVIII-a), or (XV-b), at least one R^(y) ishalo. In certain embodiments, at least one R^(y) is fluoro. In certainembodiments, at least one R^(y) is chloro. In some embodiments, at leastone R^(y) is —CN.

In some embodiments, e.g. for Formula (XV), (XVI), (XVII), (XVIII),(XV-a), (XVI-a), (XVII-a), (XVIII-a), or (XV-b), at least one R^(y) is—OR^(A), wherein R^(A) is optionally substituted aliphatic. In someembodiments, at least one R^(y) is —OR^(A), wherein R^(A) isunsubstituted C₁₋₆ alkyl. In certain embodiments, at least one R^(y) ismethoxy, ethoxy, or propoxy. In certain embodiments, at least one R^(y)is methoxy. In some embodiments, at least one R^(y) is —OR^(A), whereinR^(A) is substituted C₁₋₆ alkyl. In certain embodiments, at least oneR^(y) is —OCH₂CH₂N(CH₃)₂. In some embodiments, at least one R^(y) is—OR^(A), wherein R^(A) is optionally substituted heterocyclyl. In someembodiments, at least one R^(y) is —OR^(A), wherein R^(A) is anoptionally substituted 4- to 7-membered heterocyclyl having 1-2heteroatoms independently selected from nitrogen, oxygen, and sulfur. Insome embodiments, at least one R^(y) is —OR^(A), wherein R^(A) isoxetanyl, tetrahydrofuranyl, or tetrahydropyranyl. In some embodiments,at least one R^(y) is —OR^(A), wherein R^(A) is optionally substitutedpiperidinyl or optionally substituted piperazinyl. In some embodiments,at least one R^(y) is —OR^(A), wherein R^(A) is optionally substitutedheterocyclyl. In some embodiments, at least one R^(y) is —OR^(A),wherein R^(A) is optionally substituted heteroaryl. In some embodiments,at least one R^(y) is —OR^(A), wherein R^(A) is optionally substitutedcycloalkyl.

In some embodiments, e.g. for Formula (XV), (XVI), (XVII), (XVIII),(XV-a), (XVI-a), (XVII-a), (XVIII-a), or (XV-b), at least one R^(y) is—N(R^(B))₂. In some embodiments, at least one R^(y) is —N(R^(B))₂,wherein each R^(B) is independently selected from hydrogen or C₁₋₆alkyl. In some embodiments, at least one R^(y) is —NHR^(B). In someembodiments, at least one R^(y) is —N(C₁₋₆ alkyl)₂, —NH(C₁₋₆alkyl), or—NH₂. In certain embodiments, at least one R^(y) is —NH₂. In certainembodiments, at least one R^(y) is —NHCH₃. In certain embodiments, atleast one R^(y) is —N(CH₃)₂. In some embodiments, at least one R^(y) is—N(R^(B))₂ or —NHR^(B), wherein at least one R^(B) is optionallysubstituted heterocyclyl. In some embodiments, at least one R^(y) is—N(R^(B))₂ or —NHR^(B), wherein at least one R^(B) is an optionallysubstituted 4- to 7-membered heterocyclyl having 1-2 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, at least one R^(y) is —N(R^(B))₂ or —NHR^(B), wherein atleast one R^(B) is oxetanyl, tetrahydropyranyl, or tetrahydrofuranyl. Insome embodiments, at least one R^(y) is —N(R^(B))₂ or —NHR^(B), whereinat least one R^(B) is optionally substituted piperidinyl or optionallysubstituted piperazinyl. In some embodiments, at least one R^(y) is—N(R^(B))₂, wherein one R^(B) is optionally substituted heterocyclyl,and the other R^(B) is C₁₋₄ alkyl. In some embodiments, at least oneR^(y) is —N(R^(B))₂, wherein one R^(B) is optionally substitutedheteroaryl, and the other R^(B) is C₁₋₄ alkyl. In some embodiments, atleast one R^(y) is —N(R^(B))₂, wherein one R^(B) is optionallysubstituted cycloalkyl, and the other R^(B) is C₁₋₄ alkyl.

In some embodiments, e.g. for Formula (XV), (XVI), (XVII), (XVIII),(XV-a), (XVI-a), (XVII-a), (XVIII-a), or (XV-b), at least one R^(y) isoptionally substituted aliphatic. In certain embodiments, at least oneR^(y) is substituted aliphatic. In certain embodiments, at least oneR^(y) is unsubstituted aliphatic. In some embodiments, at least oneR^(y) is optionally substituted C₁₋₆ alkyl. In certain embodiments, atleast one R^(y) is unsubstituted C₁₋₆ alkyl. In certain embodiments, atleast one R^(y) is substituted C₁₋₆ alkyl. In certain embodiments, atleast one R^(y) is methyl, ethyl, or propyl. In certain embodiments, atleast one R^(y) is methyl. In certain embodiments, at least one

R^(y) is —CF₃, CHF₂, or CH₂F. In certain embodiments, at least one R^(y)is C₁₋₆ alkyl substituted with aryl, heteroaryl, or heterocyclyl. Incertain embodiments, at least one R^(y) is benzyl. In certainembodiments, at least one R^(y) is —(C₁₋₆ alkyl)-aryl. In certainembodiments, at least one R^(y) is —(C₁₋₆ alkyl)-heteroaryl. In certainembodiments, at least one R^(y) is —(C₁₋₆ alkyl)-heterocyclyl. Incertain embodiments, at least one R^(y) is —CH₂-aryl. In certainembodiments, at least one R^(y) is —CH₂-heteroaryl. In certainembodiments, at least one R^(y) is —CH₂-heterocyclyl.

In some embodiments, e.g. for Formula (XV), (XVI), (XVII), (XVIII),(XV-a), (XVI-a), (XVII-a), (XVIII-a), or (XV-b), at least one R^(y) is—C(O)N(R^(B))₂. In certain embodiments, at least one R^(y) is—C(O)NHR^(B). In certain embodiments, at least one R^(y) is —C(O)NH₂. Incertain embodiments, at least one R^(y) is —C(O)N(R^(B))₂, wherein theR^(B) groups are taken together with their intervening atoms to form anoptionally substituted 5- to 6-membered heterocyclyl. In certainembodiments, at least one R^(y) is —C(O)N(R^(B))₂, wherein the R^(B)groups are taken together with their intervening atoms to form anoptionally substituted morpholinyl.

In some embodiments, e.g. for Formula (XV), (XVI), (XVII), (XVIII),(XV-a), (XVI-a), (XVII-a), (XVIII-a), or (XV-b), at least one R^(y) is—SO₂N(R^(B))₂. In certain embodiments, at least one R^(y) is—SO₂NHR^(B). In certain embodiments, at least one R^(y) is —SO₂NH₂. Incertain embodiments, at least one R^(y) is —SO₂N(R^(B))₂, whereinneither R^(B) is hydrogen. In certain embodiments, at least one R^(y) is—SO₂NH(C₁₋₆ alkyl) or —SO₂N(C₁₋₆ alkyl)₂. In certain embodiments, atleast one R^(y) is —SO₂N(CH₃)₂. In certain embodiments, at least oneR^(y) is —SO₂N(R^(B))₂, wherein the R^(B) groups are taken together withtheir intervening atoms to form an optionally substituted 5- to6-membered heterocyclyl. In certain embodiments, at least one R^(y) is—SO₂-morpholinyl. In certain embodiments, at least one R^(y) is—SO₂-piperidinyl, —SO₂-piperazinyl, or —SO₂-piperidinyl.

In some embodiments, e.g. for Formula (XV), (XVI), (XVII), (XVIII),(XV-a), (XVI-a), (XVII-a), (XVIII-a), or (XV-b), at least one R^(y) is—SO₂R^(A). In some embodiments, at least one R^(y) is —SO₂R^(A), whereinR^(A) is optionally substituted aliphatic. In some embodiments, at leastone R^(y) is —SO₂(C₁₋₆ alkyl). In some embodiments, at least one R^(y)is —SO₂CH₃. In some embodiments, at least one R^(y) is —C(O)R^(A). Insome embodiments, at least one R^(y) is —C(O)R^(A), wherein R^(A) isoptionally substituted aliphatic. In some embodiments, at least oneR^(y) is —C(O)(C₁₋₆ alkyl). In some embodiments, at least one R^(y) is—C(O)CH₃.

In some embodiments, e.g. for Formula (XV), (XVI), (XVII), (XVIII),(XV-a), (XVI-a), (XVII-a), (XVIII-a), or (XV-b), at least one R^(y) is—N(R^(B))C(O)R^(A). In certain embodiments, at least one R^(y) is—NHC(O)R^(A). In certain embodiments, at least one R^(y) is —NHC(O)(C₁₋₆alkyl). In certain embodiments, at least one R^(y) is —NHC(O)CH₃.

In some embodiments, e.g. for Formula (XV), (XVI), (XVII), (XVIII),(XV-a), (XVI-a), (XVII-a), (XVIII-a), or (XV-b), at least one R^(y) is—N(R^(B))SO₂R^(A). In some embodiments, at least one R^(y) is—NHSO₂R^(A). In some embodiments, at least one R^(y) is —N(C₁₋₆alkyl)SO₂R^(A). In certain embodiments, at least one R^(y) is—NHSO₂(C₁₋₆ alkyl) or —N(C₁₋₆ alkyl)SO₂(C₁₋₆ alkyl). In certainembodiments, at least one R^(y) is —NHSO₂CH₃. In certain embodiments, atleast one R^(y) is —N(CH₃)SO₂CH₃.

In some embodiments, e.g. for Formula (XV), (XVI), (XVII), (XVIII),(XV-a), (XVI-a), (XVII-a), (XVIII-a), or (XV-b), at least one R^(y) isoptionally substituted heterocyclyl, optionally substituted carbocyclyl,optionally substituted aryl, or optionally substituted heteroaryl. Incertain embodiments, at least one R^(y) is an optionally substituted 5-to 6-membered heterocyclyl having 1-2 heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur. In certain embodiments, at least oneR^(y) is an optionally substituted 5-membered heterocyclyl having oneheteroatom selected from nitrogen, oxygen, and sulfur. In certainembodiments, at least one R^(y) is optionally substituted pyrrolidinyl.In certain embodiments, at least one R^(y) is pyrroldinyl,hydroxypyrrolidinyl, or methylpyrrolidinyl. In certain embodiments, atleast one R^(y) is an optionally substituted 6-membered heterocyclylhaving 1-2 heteroatoms independently selected from nitrogen, oxygen, andsulfur. In certain embodiments, at least one R^(y) is an optionallysubstituted 6-membered heterocyclyl having one heteroatom selected fromnitrogen, oxygen, and sulfur. In certain embodiments, at least one R^(y)is optionally substituted piperidinyl. In certain embodiments, at leastone R^(y) is an optionally substituted 6-membered heterocyclyl havingtwo heteroatoms independently selected from nitrogen, oxygen, andsulfur. In certain embodiments, at least one R^(y) is optionallysubstituted piperidinyl, optionally substituted piperazinyl, oroptionally substituted morpholinyl. In certain embodiments, at least oneR^(y) is morpholinyl, tetrahydropyranyl, piperidinyl, methylpiperidinyl,piperazinyl, methylpiperazinyl, acetylpiperazinyl,methylsulfonylpiperazinyl, aziridinyl, or methylaziridinyl. In someembodiments, at least one R^(y) is an optionally substituted 5- to6-membered heteroaryl having 1-3 heteroatoms independently selected fromnitrogen, oxygen, and sulfur. In certain embodiments, at least one R^(y)is an optionally substituted 5-membered heteroaryl having 1-3heteroatoms independently selected from nitrogen, oxygen, and sulfur. Incertain embodiments, at least one R^(y) is an optionally substituted5-membered heteroaryl having one heteroatom selected from nitrogen,oxygen, and sulfur. In certain embodiments, at least one R^(y) is anoptionally substituted 5-membered heteroaryl having two heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In certainembodiments, at least one R^(y) is an optionally substituted 6-memberedheteroaryl having 1-3 nitrogens. In certain embodiments, at least oneR^(y) is an optionally substituted pyrazolyl. In certain embodiments, atleast one R^(y) is an optionally substituted imidazolyl. In certainembodiments, at least one R^(y) is an optionally substituted pyridyl. Incertain embodiments, at least one R^(y) is an optionally substitutedpyrimidyl. In certain embodiments, at least one R^(y) is pyrazolyl,methylpyrazolyl, imidazolyl, or methylimidazolyl.

In certain embodiments, a provided compound is a compound listed inTable 1, or a pharmaceutically acceptable salt thereof.

TABLE 1 Exemplary Compounds LCMS Cmpd Exact m/z No Structure Mass (M +H)  1

387.1947 388.2  2

390.2056 391.2  3

310.1681 311.1  4

310.1681 311.1  5

325.179 326.2  6

325.179 326.2  7

326.163 327.2  8

387.1947 388.2  9

387.1947 388.2  10

376.1899 377.2  11

326.163 327.2  12

387.1947 388.2  13

387.1947 388.2  14

395.2209 396.2  15

423.2522 424.2  16

409.2365 410.2  17

311.1634 312.1  18

311.1634 312.2  19

387.1947 388.2  20

387.1947 388.2  21

389.1409 390.1  22

353.1739 354.1  23

367.1896 368.1  24

403.1566 404.1  25

353.1739 354.2  26

367.1896 368.2  27

403.1566 404.2  28

397.2365 398.1  29

408.2525 409.2  30

422.2682 423.2  31

403.1566 404.2  32

389.1409 390.1  33

389.1409 390  34

393.2416 394.1  35

394.2369 395.2  36

408.2525 409.2  37

379.226 380.2  38

393.2416 394.2  39

383.2209 384.2  40

423.2522 424.2  41

451.2835 452.3  42

379.226 380.2  43

409.2365 410.2  44

409.2365 410.2  45

395.2209 396.2  46

423.2158 424.2  47

437.2678 438.3  48

410.2206 411.2  49

423.2522 424.1  50

381.2052 382.2  51

409.2365 410.1  52

437.2678 438.3  53

437.2678 438.3  54

410.2318 411.1  55

410.2318 411.1  56

439.2471 440.1  57

427.2271 428.2  58

410.2206 411.2  59

408.2413 409.1  60

409.2365 410.2  61

438.2631 439.2  62

411.227 412.2  63

411.227 412.2  64

443.1976 444.1  65

427.2271 428  66

409.2365 410.1  67

439.2471 440.2  68

361.179 362.1  69

397.2365 398.2  70

397.2365 398.2  71

423.2522 424.2  72

383.2209 384.2  73

410.2318 411.1  74

410.2318 411.2  75

411.227 412.1  76

411.227 412.2  77

439.2471 440.2  78

427.2271 428.2  79

427.2271 428.2  80

395.2209 396.2  81

395.2209 396.2  82

410.2206 411.1  83

410.2206 411.1  84

375.1947 376  85

362.1743 363.1  86

406.2005 407.2  87

383.2209 384.2  88

367.1896 368.1  89

381.1689 382.1  90

436.2838 437.2  91

486.2301 487.2  92

490.2556 491.3  93

394.2369 395.2  94

408.2525 409.3  95

423.2522  96

409.2365 410.3  97

395.2209  98

425.2315 426.2  99

394.2256 395.2 100

450.2631 451.2 101

436.2838 437.2 102

476.2399 477.2 103

450.2995 451.3 104

409.2365 410.2 105

423.2522 424.2 106

451.2835 452.2 107

451.2471 452.2 108

487.2141 488.2 109

491.2396 492.2 110

377.1852 378.2 111

423.2522 424.2 112

376.1899 377.1 113

452.2787 453.2 114

466.2944 467.2 115

452.2787 453.2 116

396.2161 397.1 117

410.2318 411.1 118

424.2474 425.1 119

395.2209 396.2 120

408.2525 409.2 121

436.2474 437.2 122

472.2144 473 123

422.2682 423.2 124

450.2631 451.3 125

486.2301 487.2 126

490.2556 491.2 127

450.2631 451.3 128

490.2556 491.2 129

395.2209 396.2 130

377.1852 378.2 131

436.2838 437.2 132

422.2682 423.2 133

439.2471 440.2 134

409.2365 410.3 135

437.2678 438.3 136

437.2315 438.2 137

477.2239 478.3 138

408.2525 409.3 139

422.2682 423.2 140

450.2995 451.2 141

486.2301 487.2 142

396.2049 397.2 143

408.2525 409.3 144

409.2365 410.2 145

409.2365 410.2 146

398.2206 399.2 147

451.2947 452.2 148

300.1586 315.2 149

314.1743 315.1 150

314.1743 315.1 151

340.1787 341.1 152

437.2678 438.3 153

437.2678 438.3 154

380.21 381.2 155

391.1896 392.2 156

493.3053 494.2 157

466.258 467.2 158

494.2893 495.3 159

493.3053 494.2 160

452.2787 453.3 161

436.2838 437.2 162

473.1984 474.2 163

422.2682 423.3 164

443.1879 444.2 165

494.2893 495.2 166

383.1957 384.1 167

423.2522 424.2 168

423.2522 424.2 169

399.227 400.2 170

300.1586 301.1 171

314.1743 315.1 172

465.274 466.2 173

479.2896 480.3 174

493.3053 494.4 175

507.3209 508.3 176

395.2209 396.2 177

409.2365 410.2 178

411.2522 412.2 179

443.1879 444.2 180

410.243 411.2 181

410.243 411.3 182

478.2304 479.3 183

411.2158 412.3 184

410.2318 411.3 185

411.227 412.1 186

411.2634 412.3 187

380.2212 381.3 188

380.2212 381.2 189

417.2165 418.2 190

417.2165 418.3 191

417.2165 418.2 192

410.2318 411.3 193

411.227 412.2 194

521.3366 522.3 195

410.2318 411.2 196

437.2678 438.3 197

437.2315 438.2 198

473.1984 474.2 199

477.2239 478.3 200

409.2478 410.3 201

395.2321 396.2 202

424.2474 425.3 203

492.2348 493.3 204

488.2093 489.3 205

452.2424 453.3 206

424.2587 425.2 207

492.2461 493.3 208

452.2536 453.3 209

396.2274 397.3 210

438.2379 439.3 211

396.2161 397.1 212

423.2522 424.3 213

423.2522 424.3 214

397.2478 398.2 215

450.2631 451.3 216

486.2301 487.3 217

490.2556 491.3 218

361.179 362.1 219

375.1947 376.1 220

361.179 362.1 221

375.1947 376.1 222

426.2267 427.1 223

423.2634 424.1 224

491.2508 492.2 225

487.2253 488.3 226

477.2352 478.3 227

473.2097 474.2 228

437.2427 438.3 229

410.2318 411.3 230

397.2114 398.1 231

425.2427 426.1 232

425.2427 426.3 233

397.2478 398.3 234

398.2318 399.3 235

423.2634 424.3 236

423.2634 424.3 237

423.2634 424.3 238

425.2427 426.3 239

422.2682 423.1 240

349.179 350.1 241

350.1743 351.1 242

350.1743 351.1 243

352.1787 353.2 244

354.158 355 245

362.1743 363.1 246

363.1947 364.1 247

364.2151 365.1 248

366.1402 367 249

368.1736 369.1 250

380.1736 381.1 251

390.1943 391.1 252

507.3209 508.2 253

452.2787 453.2 254

451.2583 452.3 255

409.2478 410.3 256

412.2111 413.1 257

474.2049 475.3 258

411.227 412.2 259

395.2321 396.1 260

410.2318 411.1 261

425.2427 426.3 262

461.2097 462.3 263

475.2253 476.3 264

437.2791 438.3 265

439.2583 440.3 266

436.2474 437.3 267

472.2144 473.3 268

472.2144 473.3 269

349.179 350.2 270

349.179 350 271

350.163 351 272

361.179 362.1 273

367.1354 368 274

368.1736 369.1 275

379.2008 380.1 276

383.1401 384.2 277

440.2424 441.1 278

459.194 460.2 279

423.227 424.3 280

382.2117 383.1 281

396.2274 397.2 282

464.2148 465.1 283

460.1893 461.2 284

424.2223 425.3 285

493.2301 494.1 286

489.2046 490.3 287

453.2376 454.3 288

424.2474 425.3 289

492.2348 493.3 290

488.2093 489.2 291

439.2583 440.3 292

437.2791 438.3 293

436.2474 437.3 294

350.1743 351.1 295

360.1838 361.1 296

367.1696 368.2 297

488.2206 489.3 298

410.2318 411.1 299

382.2005 383.1 300

491.2508 492.1 301

487.2253 488.1 302

451.2583 452.3 303

477.2352 478.1 304

452.2424 453.3 305

351.1695 352.1 306

396.2161 397.2 307

424.2474 425.1 308

410.2318 411.1 309

425.2315 426.1 310

409.2478 410.3 311

413.2427 414.3 312

413.2427 301.1 313

439.2583 440.1 314

383.2321 384.1 315

425.2427 426.1 316

451.2195 452.3 317

361.179 362.1 318

376.1787 377.1 319

428.0848 429 320

369.2165 370.1 321

453.2628 454.2 322

493.2941 494.2 323

411.2158 412.3 324

424.2474 425.1 325

406.2117 407.3 326

448.2587 449.3 327

376.1787 377.2 328

381.2052 382.2 329

467.2784 468.2 330

499.2835 500.2 331

500.2787 501.2 332

410.2318 411.1 333

394.2369 395.3 334

394.2369 395.3 335

408.2525 409.1 336

383.1957

In certain embodiments, a provided compound inhibits PRMT5. In certainembodiments, a provided compound inhibits wild-type PRMT5. In certainembodiments, a provided compound inhibits a mutant PRMT5. In certainembodiments, a provided compound inhibits PRMT5, e.g., as measured in anassay described herein. In certain embodiments, the PRMT5 is from ahuman. In certain embodiments, a provided compound inhibits PRMT5 at anIC₅₀ less than or equal to 10 μM. In certain embodiments, a providedcompound inhibits PRMT5 at an IC₅₀ less than or equal to 1 μM. Incertain embodiments, a provided compound inhibits PRMT5 at an IC₅₀ lessthan or equal to 0.1 μM. In certain embodiments, a provided compoundinhibits PRMT5 in a cell at an EC₅₀ less than or equal to 10 μM. Incertain embodiments, a provided compound inhibits PRMT5 in a cell at anEC₅₀ less than or equal to 1 μM. In certain embodiments, a providedcompound inhibits PRMT5 in a cell at an EC₅₀ less than or equal to 0.1μM. In certain embodiments, a provided compound inhibits cellproliferation at an EC₅₀ less than or equal to 10 μM. In certainembodiments, a provided compound inhibits cell proliferation at an EC₅₀less than or equal to 1 μM. In certain embodiments, a provided compoundinhibits cell proliferation at an EC₅₀ less than or equal to 0.1 μM. Insome embodiments, a provided compound is selective for PRMT5 over othermethyltransferases. In certain embodiments, a provided compound is atleast about 10-fold selective, at least about 20-fold selective, atleast about 30-fold selective, at least about 40-fold selective, atleast about 50-fold selective, at least about 60-fold selective, atleast about 70-fold selective, at least about 80-fold selective, atleast about 90-fold selective, or at least about 100-fold selective forPRMT5 relative to one or more other methyltransferases.

It will be understood by one of ordinary skill in the art that the PRMT5can be wild-type PRMT5, or any mutant or variant of PRMT5.

In certain embodiments, the PRMT5 is isoform A (GenBank accession no.NP006100) (SEQ ID NO.:1):

MAAMAVGGAG GSRVSSGRDL NCVPEIADTL GAVAKQGFDFLCMPVFHPRF KREFIQEPAK NRPGPQTRSD LLLSGRDWNTLIVGKLSPWI RPDSKVEKIR RNSEAAMLQE LNFGAYLGLPAFLLPLNQED NTNLARVLTN HIHTGHHSSM FWMRVPLVAPEDLRDDIIEN APTTHTEEYS GEEKTWMWWH NFRTLCDYSKRIAVALEIGA DLPSNHVIDR WLGEPIKAAI LPTSIFLTNKKGFPVLSKMH QRLIFRLLKL EVQFIITGTN HHSEKEFCSYLQYLEYLSQN RPPPNAYELF AKGYEDYLQS PLQPLMDNLESQTYEVFEKD PIKYSQYQQA IYKCLLDRVP EEEKDTNVQVLMVLGAGRGP LVNASLRAAK QADRRIKLYA VEKNPNAVVTLENWQFEEWG SQVTVVSSDM REWVAPEKAD IIVSELLGSFADNELSPECL DGAQHFLKDD GVSIPGEYTS FLAPISSSKLYNEVRACREK DRDPEAQFEM PYVVRLHNFH QLSAPQPCFTFSHPNRDPMI DNNRYCTLEF PVEVNTVLHG FAGYFETVLYQDITLSIRPE THSPGMFSWF PILFPIKQPI TVREGQTICVRFWRCSNSKK VWYEWAVTAP VCSAIHNPTG RSYTIGL

In certain embodiments, the PRMT5 is isoform B (GenBank accession no.NP001034708) (SEQ ID NO.:2)

MRGPNSGTEK GRLVIPEKQG FDFLCMPVFH PRFKREFIQEPAKNRPGPQT RSDLLLSGRD WNTLIVGKLS PWIRPDSKVEKIRRNSEAAM LQELNFGAYL GLPAFLLPLN QEDNTNLARVLTNHIHTGHH SSMFWMRVPL VAPEDLRDDI IENAPTTHTE EYSGEEKTWM WWHNFRTLCD YSKRIAVALE IGADLPSNHVIDRWLGEPIK AAILPTSIFL TNKKGFPVLS KMHQRLIFRLLKLEVQFIIT GTNHHSEKEF CSYLQYLEYL SQNRPPPNAYELFAKGYEDY LQSPLQPLMD NLESQTYEVF EKDPIKYSQYQQAIYKCLLD RVPEEEKDTN VQVLMVLGAG RGPLVNASLRAAKQADRRIK LYAVEKNPNA VVTLENWQFE EWGSQVTVVSSDMREWVAPE KADIIVSELL GSFADNELSP ECLDGAQHFLKDDGVSIPGE YTSFLAPISS SKLYNEVRAC REKDRDPEAQFEMPYVVRLH NFHQLSAPQP CFTFSHPNRD PMIDNNRYCTLEFPVEVNTV LHGFAGYFET VLYQDITLSI RPETHSPGMFSWFPILFPIK QPITVREGQT ICVRFWRCSN SKKVWYEWAV TAPVCSAIHN PTGRSYTIGL

In certain embodiments, the PRMT5 is transcript variant 1 (GenBankaccession no. NM_(—)006109).

The present disclosure provides pharmaceutical compositions comprising acompound described herein, e.g., a compound of Formula (I), or apharmaceutically acceptable salt thereof, as described herein, andoptionally a pharmaceutically acceptable excipient. It will beunderstood by one of ordinary skill in the art that the compoundsdescribed herein, or salts thereof, may be present in various forms,such as amorphous, hydrates, solvates, or polymorphs. In certainembodiments, a provided composition comprises two or more compoundsdescribed herein. In certain embodiments, a compound described herein,or a pharmaceutically acceptable salt thereof, is provided in aneffective amount in the pharmaceutical composition. In certainembodiments, the effective amount is a therapeutically effective amount.In certain embodiments, the effective amount is an amount effective forinhibiting PRMT5. In certain embodiments, the effective amount is anamount effective for treating a PRMT5-mediated disorder. In certainembodiments, the effective amount is a prophylactically effectiveamount. In certain embodiments, the effective amount is an amounteffective to prevent a PRMT5-mediated disorder.

Pharmaceutically acceptable excipients include any and all solvents,diluents, or other liquid vehicles, dispersions, suspension aids,surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, solid binders, lubricants, and the like, assuited to the particular dosage form desired. General considerations informulation and/or manufacture of pharmaceutical compositions agents canbe found, for example, in Remington's Pharmaceutical Sciences, SixteenthEdition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), andRemington: The Science and Practice of Pharmacy, 21st Edition(Lippincott Williams & Wilkins, 2005).

Pharmaceutical compositions described herein can be prepared by anymethod known in the art of pharmacology. In general, such preparatorymethods include the steps of bringing a compound described herein (the“active ingredient”) into association with a carrier and/or one or moreother accessory ingredients, and then, if necessary and/or desirable,shaping and/or packaging the product into a desired single- ormulti-dose unit.

Pharmaceutical compositions can be prepared, packaged, and/or sold inbulk, as a single unit dose, and/or as a plurality of single unit doses.As used herein, a “unit dose” is discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient which would be administered to a subject and/or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition of the present disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100% (w/w) active ingredient.

Pharmaceutically acceptable excipients used in the manufacture ofprovided pharmaceutical compositions include inert diluents, dispersingand/or granulating agents, surface active agents and/or emulsifiers,disintegrating agents, binding agents, preservatives, buffering agents,lubricating agents, and/or oils. Excipients such as cocoa butter andsuppository waxes, coloring agents, coating agents, sweetening,flavoring, and perfuming agents may also be present in the composition.

Exemplary diluents include calcium carbonate, sodium carbonate, calciumphosphate, dicalcium phosphate, calcium sulfate, calcium hydrogenphosphate, sodium phosphate lactose, sucrose, cellulose,microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodiumchloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.

Exemplary granulating and/or dispersing agents include potato starch,corn starch, tapioca starch, sodium starch glycolate, clays, alginicacid, guar gum, citrus pulp, agar, bentonite, cellulose and woodproducts, natural sponge, cation-exchange resins, calcium carbonate,silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone)(crospovidone), sodium carboxymethyl starch (sodium starch glycolate),carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose(croscarmellose), methylcellulose, pregelatinized starch (starch 1500),microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate,quaternary ammonium compounds, and mixtures thereof.

Exemplary surface active agents and/or emulsifiers include naturalemulsifiers (e.g., acacia, agar, alginic acid, sodium alginate,tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk,casein, wool fat, cholesterol, wax, and lecithin), colloidal clays(e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminumsilicate)), long chain amino acid derivatives, high molecular weightalcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetinmonostearate, ethylene glycol distearate, glyceryl monostearate, andpropylene glycol monostearate, polyvinyl alcohol), carbomers (e.g.,carboxy polymethylene, polyacrylic acid, acrylic acid polymer, andcarboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.,carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylenesorbitan monolaurate (Tween 20), polyoxyethylene sorbitan (Tween 60),polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monopalmitate(Span 40), sorbitan monostearate (Span 60], sorbitan tristearate (Span65), glyceryl monooleate, sorbitan monooleate (Span 80)),polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj 45),polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and Solutol), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g., Cremophor™),polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (Brij 30)),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, Pluronic F68, Poloxamer 188, cetrimoniumbromide, cetylpyridinium chloride, benzalkonium chloride, docusatesodium, and/or mixtures thereof.

Exemplary binding agents include starch (e.g., cornstarch and starchpaste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin,molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums(e.g., acacia, sodium alginate, extract of Irish moss, panwar gum,ghatti gum, mucilage of isapol husks, carboxymethylcellulose,methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, microcrystalline cellulose,cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate(Veegum), and larch arabogalactan), alginates, polyethylene oxide,polyethylene glycol, inorganic calcium salts, silicic acid,polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.

Exemplary preservatives include antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, alcoholpreservatives, acidic preservatives, and other preservatives.

Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene,monothioglycerol, potassium metabisulfite, propionic acid, propylgallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, andsodium sulfite.

Exemplary chelating agents include ethylenediaminetetraacetic acid(EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodiumedetate, trisodium edetate, calcium disodium edetate, dipotassiumedetate, and the like), citric acid and salts and hydrates thereof(e.g., citric acid monohydrate), fumaric acid and salts and hydratesthereof, malic acid and salts and hydrates thereof, phosphoric acid andsalts and hydrates thereof, and tartaric acid and salts and hydratesthereof. Exemplary antimicrobial preservatives include benzalkoniumchloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide,cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol,chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea,phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate,propylene glycol, and thimerosal.

Exemplary antifungal preservatives include butyl paraben, methylparaben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoicacid, potassium benzoate, potassium sorbate, sodium benzoate, sodiumpropionate, and sorbic acid.

Exemplary alcohol preservatives include ethanol, polyethylene glycol,phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate,and phenylethyl alcohol. Exemplary acidic preservatives include vitaminA, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid,dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopherol acetate, deteroximemesylate, cetrimide, butylated hydroxyanisol (BHA), butylatedhydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS),sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus,Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, andEuxyl. In certain embodiments, the preservative is an anti-oxidant. Inother embodiments, the preservative is a chelating agent.

Exemplary buffering agents include citrate buffer solutions, acetatebuffer solutions, phosphate buffer solutions, ammonium chloride, calciumcarbonate, calcium chloride, calcium citrate, calcium glubionate,calcium gluceptate, calcium gluconate, D-gluconic acid, calciumglycerophosphate, calcium lactate, propanoic acid, calcium levulinate,pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasiccalcium phosphate, calcium hydroxide phosphate, potassium acetate,potassium chloride, potassium gluconate, potassium mixtures, dibasicpotassium phosphate, monobasic potassium phosphate, potassium phosphatemixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodiumcitrate, sodium lactate, dibasic sodium phosphate, monobasic sodiumphosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide,aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline,Ringer's solution, ethyl alcohol, and mixtures thereof.

Exemplary lubricating agents include magnesium stearate, calciumstearate, stearic acid, silica, talc, malt, glyceryl behanate,hydrogenated vegetable oils, polyethylene glycol, sodium benzoate,sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate,sodium lauryl sulfate, and mixtures thereof.

Exemplary natural oils include almond, apricot kernel, avocado, babassu,bergamot, black current seed, borage, cade, camomile, canola, caraway,carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee,corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed,geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate,jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademianut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed,pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, andwheat germ oils. Exemplary synthetic oils include, but are not limitedto, butyl stearate, caprylic triglyceride, capric triglyceride,cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate,mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixturesthereof.

Liquid dosage forms for oral and parenteral administration includepharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active ingredients,the liquid dosage forms may comprise inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can include adjuvants such as wetting agents, emulsifyingand suspending agents, sweetening, flavoring, and perfuming agents. Incertain embodiments for parenteral administration, the compoundsdescribed herein are mixed with solubilizing agents such as Cremophor™,alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins,polymers, and mixtures thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation can be a sterile injectable solution,suspension or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that can be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This can be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing the compounds describedherein with suitable non-irritating excipients or carriers such as cocoabutter, polyethylene glycol or a suppository wax which are solid atambient temperature but liquid at body temperature and therefore melt inthe rectum or vaginal cavity and release the active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may comprise buffering agents.

Solid compositions of a similar type can be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type can be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

The active ingredient can be in micro-encapsulated form with one or moreexcipients as noted above. The solid dosage forms of tablets, dragees,capsules, pills, and granules can be prepared with coatings and shellssuch as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active ingredient can be admixed with at least oneinert diluent such as sucrose, lactose, or starch. Such dosage forms maycomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets, and pills, the dosage forms may comprise bufferingagents. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Dosage forms for topical and/or transdermal administration of a providedcompound may include ointments, pastes, creams, lotions, gels, powders,solutions, sprays, inhalants and/or patches. Generally, the activeingredient is admixed under sterile conditions with a pharmaceuticallyacceptable carrier and/or any desired preservatives and/or buffers ascan be required. Additionally, the present disclosure encompasses theuse of transdermal patches, which often have the added advantage ofproviding controlled delivery of an active ingredient to the body. Suchdosage forms can be prepared, for example, by dissolving and/ordispensing the active ingredient in the proper medium. Alternatively oradditionally, the rate can be controlled by either providing a ratecontrolling membrane and/or by dispersing the active ingredient in apolymer matrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositionscan be administered by devices which limit the effective penetrationlength of a needle into the skin, such as those described in PCTpublication WO 99/34850 and functional equivalents thereof. Jetinjection devices which deliver liquid vaccines to the dermis via aliquid jet injector and/or via a needle which pierces the stratumcorneum and produces a jet which reaches the dermis are suitable. Jetinjection devices are described, for example, in U.S. Pat. Nos.5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189;5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335;5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880;4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis are suitable. Alternatively or additionally, conventionalsyringes can be used in the classical mantoux method of intradermaladministration.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient can be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A provided pharmaceutical composition can be prepared, packaged, and/orsold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers or from about 1 to about 6nanometers. Such compositions are conveniently in the form of drypowders for administration using a device comprising a dry powderreservoir to which a stream of propellant can be directed to dispersethe powder and/or using a self propelling solvent/powder dispensingcontainer such as a device comprising the active ingredient dissolvedand/or suspended in a low-boiling propellant in a sealed container. Suchpowders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositions mayinclude a solid fine powder diluent such as sugar and are convenientlyprovided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic and/or solid anionic surfactant and/or a solid diluent(which may have a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions formulated for pulmonary delivery mayprovide the active ingredient in the form of droplets of a solutionand/or suspension. Such formulations can be prepared, packaged, and/orsold as aqueous and/or dilute alcoholic solutions and/or suspensions,optionally sterile, comprising the active ingredient, and mayconveniently be administered using any nebulization and/or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, and/or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration may have an average diameter inthe range from about 0.1 to about 200 nanometers.

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition. Anotherformulation suitable for intranasal administration is a coarse powdercomprising the active ingredient and having an average particle fromabout 0.2 to 500 micrometers. Such a formulation is administered byrapid inhalation through the nasal passage from a container of thepowder held close to the nares.

Formulations for nasal administration may, for example, comprise fromabout as little as 0.1% (w/w) and as much as 100% (w/w) of the activeingredient, and may comprise one or more of the additional ingredientsdescribed herein. A provided pharmaceutical composition can be prepared,packaged, and/or sold in a formulation for buccal administration. Suchformulations may, for example, be in the form of tablets and/or lozengesmade using conventional methods, and may contain, for example, 0.1 to20% (w/w) active ingredient, the balance comprising an orallydissolvable and/or degradable composition and, optionally, one or moreof the additional ingredients described herein. Alternately,formulations for buccal administration may comprise a powder and/or anaerosolized and/or atomized solution and/or suspension comprising theactive ingredient. Such powdered, aerosolized, and/or aerosolizedformulations, when dispersed, may have an average particle and/ordroplet size in the range from about 0.1 to about 200 nanometers, andmay further comprise one or more of the additional ingredients describedherein.

A provided pharmaceutical composition can be prepared, packaged, and/orsold in a formulation for ophthalmic administration. Such formulationsmay, for example, be in the form of eye drops including, for example, a0.1/1.0% (w/w) solution and/or suspension of the active ingredient in anaqueous or oily liquid carrier. Such drops may further comprisebuffering agents, salts, and/or one or more other of the additionalingredients described herein. Other opthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form and/or in a liposomal preparation.Ear drops and/or eye drops are contemplated as being within the scope ofthis disclosure.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with ordinary experimentation.

Compounds provided herein are typically formulated in dosage unit formfor ease of administration and uniformity of dosage. It will beunderstood, however, that the total daily usage of provided compositionswill be decided by the attending physician within the scope of soundmedical judgment. The specific therapeutically effective dose level forany particular subject or organism will depend upon a variety of factorsincluding the disease, disorder, or condition being treated and theseverity of the disorder; the activity of the specific active ingredientemployed; the specific composition employed; the age, body weight,general health, sex and diet of the subject; the time of administration,route of administration, and rate of excretion of the specific activeingredient employed; the duration of the treatment; drugs used incombination or coincidental with the specific active ingredientemployed; and like factors well known in the medical arts.

The compounds and compositions provided herein can be administered byany route, including enteral (e.g., oral), parenteral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,subcutaneous, intraventricular, transdermal, interdermal, rectal,intravaginal, intraperitoneal, topical (as by powders, ointments,creams, and/or drops), mucosal, nasal, bucal, sublingual; byintratracheal instillation, bronchial instillation, and/or inhalation;and/or as an oral spray, nasal spray, and/or aerosol. Specificallycontemplated routes are oral administration, intravenous administration(e.g., systemic intravenous injection), regional administration viablood and/or lymph supply, and/or direct administration to an affectedsite. In general the most appropriate route of administration willdepend upon a variety of factors including the nature of the agent(e.g., its stability in the environment of the gastrointestinal tract),and/or the condition of the subject (e.g., whether the subject is ableto tolerate oral administration).

The exact amount of a compound required to achieve an effective amountwill vary from subject to subject, depending, for example, on species,age, and general condition of a subject, severity of the side effects ordisorder, identity of the particular compound(s), mode ofadministration, and the like. The desired dosage can be delivered threetimes a day, two times a day, once a day, every other day, every thirdday, every week, every two weeks, every three weeks, or every fourweeks. In certain embodiments, the desired dosage can be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations).

In certain embodiments, an effective amount of a compound foradministration one or more times a day to a 70 kg adult human maycomprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg,about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about1000 mg, or about 100 mg to about 1000 mg, of a compound per unit dosageform.

In certain embodiments, a compound described herein may be administeredat dosage levels sufficient to deliver from about 0.001 mg/kg to about1000 mg/kg, from about 0.01 mg/kg to about mg/kg, from about 0.1 mg/kgto about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, orfrom about 1 mg/kg to about 25 mg/kg, of subject body weight per day,one or more times a day, to obtain the desired therapeutic effect.

In some embodiments, a compound described herein is administered one ormore times per day, for multiple days. In some embodiments, the dosingregimen is continued for days, weeks, months, or years.

It will be appreciated that dose ranges as described herein provideguidance for the administration of provided pharmaceutical compositionsto an adult. The amount to be administered to, for example, a child oran adolescent can be determined by a medical practitioner or personskilled in the art and can be lower or the same as that administered toan adult.

It will be also appreciated that a compound or composition, as describedherein, can be administered in combination with one or more additionaltherapeutically active agents. In certain embodiments, a compound orcomposition provided herein is administered in combination with one ormore additional therapeutically active agents that improve itsbioavailability, reduce and/or modify its metabolism, inhibit itsexcretion, and/or modify its distribution within the body. It will alsobe appreciated that the therapy employed may achieve a desired effectfor the same disorder, and/or it may achieve different effects.

The compound or composition can be administered concurrently with, priorto, or subsequent to, one or more additional therapeutically activeagents. In certain embodiments, the additional therapeutically activeagent is a compound of Formula (I). In certain embodiments, theadditional therapeutically active agent is not a compound of Formula(I). In general, each agent will be administered at a dose and/or on atime schedule determined for that agent. In will further be appreciatedthat the additional therapeutically active agent utilized in thiscombination can be administered together in a single composition oradministered separately in different compositions. The particularcombination to employ in a regimen will take into account compatibilityof a provided compound with the additional therapeutically active agentand/or the desired therapeutic effect to be achieved. In general, it isexpected that additional therapeutically active agents utilized incombination be utilized at levels that do not exceed the levels at whichthey are utilized individually. In some embodiments, the levels utilizedin combination will be lower than those utilized individually.

Exemplary additional therapeutically active agents include, but are notlimited to, small organic molecules such as drug compounds (e.g.,compounds approved by the U.S. Food and Drug Administration as providedin the Code of Federal Regulations (CFR)), peptides, proteins,carbohydrates, monosaccharides, oligosaccharides, polysaccharides,nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides orproteins, small molecules linked to proteins, glycoproteins, steroids,nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides,antisense oligonucleotides, lipids, hormones, vitamins, and cells.

Also encompassed by the present disclosure are kits (e.g.,pharmaceutical packs). The kits provided may comprise a providedpharmaceutical composition or compound and a container (e.g., a vial,ampule, bottle, syringe, and/or dispenser package, or other suitablecontainer). In some embodiments, provided kits may optionally furtherinclude a second container comprising a pharmaceutical excipient fordilution or suspension of a provided pharmaceutical composition orcompound. In some embodiments, a provided pharmaceutical composition orcompound provided in the container and the second container are combinedto form one unit dosage form. In some embodiments, a provided kitsfurther includes instructions for use.

Compounds and compositions described herein are generally useful for theinhibition of PRMT5. In some embodiments, methods of treatingPRMT5-mediated disorder in a subject are provided which compriseadministering an effective amount of a compound described herein (e.g.,a compound of Formula (I)), or a pharmaceutically acceptable saltthereof), to a subject in need of treatment. In certain embodiments, theeffective amount is a therapeutically effective amount. In certainembodiments, the effective amount is a prophylactically effectiveamount. In certain embodiments, the subject is suffering from aPRMT5-mediated disorder. In certain embodiments, the subject issusceptible to a PRMT5-mediated disorder.

As used herein, the term “PRMT5-mediated disorder” means any disease,disorder, or other pathological condition in which PRMT5 is known toplay a role. Accordingly, in some embodiments, the present disclosurerelates to treating or lessening the severity of one or more diseases inwhich PRMT5 is known to play a role.

In some embodiments, the present disclosure provides a method ofinhibiting PRMT5 comprising contacting PRMT5 with an effective amount ofa compound described herein (e.g., a compound of Formula (I)), or apharmaceutically acceptable salt thereof. The PRMT5 may be purified orcrude, and may be present in a cell, tissue, or subject. Thus, suchmethods encompass both inhibition of in vitro and in vivo PRMT5activity. In certain embodiments, the method is an in vitro method,e.g., such as an assay method. It will be understood by one of ordinaryskill in the art that inhibition of PRMT5 does not necessarily requirethat all of the PRMT5 be occupied by an inhibitor at once. Exemplarylevels of inhibition of PRMT5 include at least 10% inhibition, about 10%to about 25% inhibition, about 25% to about 50% inhibition, about 50% toabout 75% inhibition, at least 50% inhibition, at least 75% inhibition,about 80% inhibition, about 90% inhibition, and greater than 90%inhibition.

In some embodiments, provided is a method of inhibiting PRMT5 activityin a subject in need thereof comprising administering to the subject aneffective amount of a compound described herein (e.g., a compound ofFormula (I)), or a pharmaceutically acceptable salt thereof, or apharmaceutical composition thereof.

In certain embodiments, provided is a method of altering gene expressionin a cell which comprises contacting a cell with an effective amount ofa compound of Formula (I), or a pharmaceutically acceptable saltthereof. In certain embodiments, the cell in culture in vitro. Incertain embodiments, the cell is in an animal, e.g., a human. In certainembodiments, the cell is in a subject in need of treatment.

In certain embodiments, provided is a method of altering transcriptionin a cell which comprises contacting a cell with an effective amount ofa compound of Formula (I), or a pharmaceutically acceptable saltthereof. In certain embodiments, the cell in culture in vitro. Incertain embodiments, the cell is in an animal, e.g., a human. In certainembodiments, the cell is in a subject in need of treatment.

In certain embodiments, a method is provided of selecting a therapy fora subject having a disease associated with PRMT5-mediated disorder ormutation comprising the steps of determining the presence ofPRMT5-mediated disorder or gene mutation in the PRMT5 gene or andselecting, based on the presence of PRMT5-mediated disorder a genemutation in the PRMT5 gene a therapy that includes the administration ofa provided compound. In certain embodiments, the disease is cancer.

In certain embodiments, a method of treatment is provided for a subjectin need thereof comprising the steps of determining the presence ofPRMT5-mediated disorder or a gene mutation in the PRMT5 gene andtreating the subject in need thereof, based on the presence of aPRMT5-mediated disorder or gene mutation in the PRMT5 gene with atherapy that includes the administration of a provided compound. Incertain embodiments, the subject is a cancer patient.

In some embodiments, a provided compound is useful in treating aproliferative disorder, such as cancer, a benign neoplasm, an autoimmunedisease, or an inflammatory disease. For example, while not being boundto any particular mechanism, PRMT5 has been shown to be involved incyclin D1 dysregulated cancers. Increased PRMT5 activity mediates keyevents associated with cyclin D1-dependent neoplastic growth includingCUL4 repression, CDT1 overexpression, and DNA re-replication. Further,human cancers harboring mutations in Fbx4, the cyclin D1 E3 ligase,exhibit nuclear cyclin D1 accumulation and increased PRMT5 activity(Aggarwal et al., Cancer Cell. 2010 18(4):329-40). Additionally, PRMT5has also been implicated in accelerating cell cycle progression throughG1 phase and modulating regulators of G1; for example, PRMT5 mayupregulate cyclin-dependent kinase (CDK) 4, CDK6, and cyclins D1, D2 andE1. Moreover, PRMT5 may activate phosphoinositide 3-kinase (PI3K)/AKTsignaling (Wei et al., Cancer Sci. 2012 103(9):1640-50). Thus in someembodiments, the inhibition of PRMT5 by a provided compound is useful intreating the following non-limiting list of cancers: breast cancer,esophageal cancer, bladder cancer, lung cancer, hematopoietic cancer,lymphoma, medulloblastoma, rectum adenocarcinoma, colon adenocarcinoma,gastric cancer, pancreatic cancer, liver cancer, adenoid cysticcarcinoma, lung adenocarcinoma, head and neck squamous cell carcinoma,brain tumors, hepatocellular carcinoma, renal cell carcinoma, melanoma,oligodendroglioma, ovarian clear cell carcinoma, and ovarian serouscystadenocarcinoma.

In some embodiments, the inhibition of PRMT5 by a provided compound isuseful in treating prostate cancer and lung cancer, in which PRMT5 hasbeen shown to play a role (Gu et al., PLoS One 2012; 7(8):e44033; Gu etal., Biochem. J. (2012) 446 (235-241)). In some embodiments, a providedcompound is useful to delay the onset of, slow the progression of, orameliorate the symptoms of cancer. In some embodiments, a providedcompound is administered in combination with other compounds, drugs, ortherapeutics to treat cancer.

In some embodiments, compounds described herein are useful for treatinga cancer including, but not limited to, acoustic neuroma,adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g.,lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma),appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g.,cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinomaof the breast, papillary carcinoma of the breast, mammary cancer,medullary carcinoma of the breast), brain cancer (e.g., meningioma;glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchuscancer, carcinoid tumor, cervical cancer (e.g., cervicaladenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma,colorectal cancer (e.g., colon cancer, rectal cancer, colorectaladenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma(e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma),endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophagealcancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma),Ewing sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma),familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g.,stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head andneck cancer (e.g., head and neck squamous cell carcinoma, oral cancer(e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g.,laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer,oropharyngeal cancer)), hematopoietic cancers (e.g., leukemia such asacute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acutemyelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronicmyelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chroniclymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma suchas Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkinlymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma(DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicularlymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas(e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodalmarginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma),primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacyticlymphoma (i.e., “Waldenstrom's macroglobulinemia”), hairy cell leukemia(HCL), immunoblastic large cell lymphoma, precursor B-lymphoblasticlymphoma and primary central nervous system (CNS) lymphoma; and T-cellNHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheralT-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g.,mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma,extranodal natural killer T-cell lymphoma, enteropathy type T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplasticlarge cell lymphoma); a mixture of one or more leukemia/lymphoma asdescribed above; and multiple myeloma (MM)), heavy chain disease (e.g.,alpha chain disease, gamma chain disease, mu chain disease),hemangioblastoma, inflammatory myofibroblastic tumors, immunocyticamyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor,renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC),malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, smallcell lung cancer (SCLC), non-small cell lung cancer (NSCLC),adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g.,systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma,myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV),essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a.myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocyticleukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilicsyndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis(NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g.,gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoidtumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarianembryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma,pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductalpapillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer(e.g., Paget's disease of the penis and scrotum), pinealoma, primitiveneuroectodermal tumor (PNT), prostate cancer (e.g., prostateadenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer,skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA),melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g.,appendix cancer), soft tissue sarcoma (e.g., malignant fibroushistiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor(MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous glandcarcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g.,seminoma, testicular embryonal carcinoma), thyroid cancer (e.g.,papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC),medullary thyroid cancer), urethral cancer, vaginal cancer and vulvarcancer (e.g., Paget's disease of the vulva).

In some embodiments, a provided compound is useful in treating ametabolic disorder, such as diabetes or obesity. For example, while notbeing bound to any particular mechanism, a role for PRMT5 has beenrecognized in adipogenesis. Inhibition of PRMT5 expression in multiplecell culture models for adipogenesis prevented the activation ofadipogenic genes, while overexpression of PRMT5 enhanced adipogenic geneexpression and differentiation (LeBlanc et al., Mol Endocrinol. 2012April; 26(4):583-97). Additionally, it has been shown that adipogenesisplays a pivotal role in the etiology and progression of diabetes andobesity (Camp et al., Trends Mol Med. 2002 September; 8(9):442-7). Thusin some embodiments, the inhibition of PRMT5 by a provided compound isuseful in treating diabetes and/or obesity.

In some embodiments, a provided compound is useful to delay the onsetof, slow the progression of, or ameliorate the symptoms of, diabetes. Insome embodiments, the diabetes is Type 1 diabetes. In some embodiments,the diabetes is Type 2 diabetes. In some embodiments, a providedcompound is useful to delay the onset of, slow the progression of, orameliorate the symptoms of, obesity. In some embodiments, a providedcompound is useful to make a subject lose weight. In some embodiments, aprovided compound could be used in combination with other compounds,drugs, or therapeutics, such as metformin and insulin, to treat diabetesand/or obesity.

In some embodiments, a provided compound is useful in treating a blooddisorder, e.g., a hemoglobinopathy, such as sickle cell disease orβ-thalassemia. For example, while not being bound to any particularmechanism, PRMT5 is a known repressor of γ-globin gene expression, andincreased fetal y-globin (HbF) levels in adulthood are associated withsymptomatic amelioration in sickle cell disease and β-thalassemia (Xu etal., Haematologica. 2012 November; 97(11):1632-40). Thus in someembodiments, the inhibition of PRMT5 by a provided compound is useful intreating a blood disorder, such as a hemoglobinopathy such as sicklecell disease or β-thalassemia.

In some embodiments, a provided compound is useful to delay the onsetof, slow the progression of, or ameliorate the symptoms of, sickle celldisease. In some embodiments, a provided compound is useful to delay theonset of, slow the progression of, or ameliorate the symptoms of,β-thalassemia. In some embodiments, a provided compound could be used incombination with other compounds, drugs, or therapeutics, to treat ahemoglobinopathy such as sickle cell disease or β-thalassemia.

In some embodiments, compounds described herein can prepared usingmethods shown in general Scheme 1. Compound B can be prepared via ringopening of a chiral or racemic epoxide group. This amino alcoholintermediate can be coupled to form an amide via normal amide couplingmethodology using a carboxylic acid A wherein Z is hydrogen or viaamination of an ester of intermediate A when Z is an optionallysubstituted aliphatic group.

Analogous reactions may be performed to form a carbamate or urea bondusing methods known to one of ordinary skill in the art.

In some embodiments, such couplings can be used to provide a keyintermediate for further synthesis, as shown, for example, in exemplaryScheme 2.

In other embodiments, an amide coupling step is the final synthetic stepas shown in exemplary Scheme 3.

EXAMPLES

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

Synthetic Methods Compound 1N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(pyridin-2-yl)benzamide

Step 1: methyl 3-(pyridin-2-yl)benzoate

A mixture of (3-(methoxycarbonyl)phenyl)boronic acid (500 mg, 2.78mmol), 2-bromopyridine (399 mg, 2.53 mmol), K₂CO₃ (1.0 g, 7.6 mmol) andPd(dppf)Cl₂ (20 mg) in a mixture solution of dioxane (10 mL) and H₂O(2.5 mL) was stirred at 120° C. for 30 min under microwave heating. Thecatalyst was removed by filtration and the filtrate was concentrated.The residue was purified by column chromatography to give the desiredproduct (530 mg, Yield: 90%) and this was used directly in the nextstep. LCMS (m/z): 214.1.

Step 2: 3-(pyridin-2-yl)benzoic acid

To a solution of methyl 3-(pyridin-2-yl)benzoate (300 mg, 1.40 mmol) inMeOH (3 mL) was added aqueous NaOH (1 mL, 0.4M). The mixture was stirredat room temperature for 3 h. The reaction solution was concentrated andthe residue dissolved in water and adjust pH to 5-6 with 2N of HCl. Thesolution was extracted with EtOAc (3×20 mL) and the combined organiclayers concentrated to give the desired crude product (450 mg, Yield90%) which was used in the next step without further purification. LCMS(m/z): 200.1 (M+1).

Step 3:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(pyridin-2-yl)benzamide

To a solution of 3-(pyridin-2-yl)benzoic acid (200 mg, 1.00 mmol) in DCM(6 mL) was added EDCI (383 mg, 2.00 mmol), HOBt (270 mg, 2 mmol), Et₃N(303 mg, 3 mmol) and1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (206 mg, 1.00mmol). The mixture was stirred at room temperature for 16 h. Thereaction mixture was diluted with water (10 mL) and extracted with DCM(3×10 mL). The combined organic layers were then dried and concentrated.The residue was purified by Prep-HPLC to give the product as the formatesalt (70 mg, Yield 18%). ¹H NMR (400 MHz, MeOD): 8.64 (d, J=4.8 Hz, 1H),8.46 (s, 1H), 8.13 (d, J=8.4 Hz, 1H), 7.93-7.90 (m. 3H), 7.60 (dd, J=8.0Hz, 1H), 7.40-7.37 (m, 1H), 7.26-7.14 (m, 4H), 4.44 (s, 2H), 4.38 (br.s,1H), 3.57-3.56 (m, 4H), 3.36-3.16 (m, 4H). LCMS (m/z): 388.2 (M+1).

Compound 2N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(1-methyl-1H-pyrazol-5-yl)benzamide

Step 1: methyl 3-(1-methyl-1H-pyrazol-5-yl)benzoate

A mixture of (3-(methoxycarbonyl)phenyl)boronic acid (270 mg, 1.5 mmol),5-bromo-1-methyl-1H-pyrazole (200 mg, 1.25 mmol), K₂CO₃ (518 mg, 3.75mmol) and Pd(dppf)Cl₂ (10 mg) in a mixture solution of dioxane (8 mL)and H₂O (2 mL) was stirred at 120° C. for 30 min under microwaveheating. The catalyst was filtered and the filtrate concentrated. Theresidue was then purified by column chromatography to give provide thedesired product as a colorless oil (226 mg, Yield 60%). It was useddirectly in the next step. LCMS (m/z): 217.1.

Step 2: 3-(1-methyl-1H-pyrazol-5-yl)benzoic acid

To a solution of methyl 3-(1-methyl-1H-pyrazol-5-yl)benzoate (200 mg,0.93 mmol) in MeOH (3 mL) was added aqueous NaOH (1 mL, 0.4M). Themixture was stirred at room temperature for 2 h. The reaction solutionwas concentrated and the residue was dissolved in water and adjusted pHto 5-6 with 2N of HCl. The solution was extracted with EtOAc (2×20 mL).The combined organic layers were dried and concentrated to give thetarget crude product which was used directly in the next step. LCMS(m/z): 203.1 (M+1).

Step 3:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(1-methyl-1H-pyrazol-5-yl)benzamide

To a solution of 3-(1-methyl-1H-pyrazol-5-yl)benzoic acid (130 mg, 0.64mmol) in DCM (6 mL) was added EDCI (245 mg, 1.28 mmol), HOBt (173 mg,1.28 mmol), Et₃N (195 mg, 1.93 mmol) and1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (132 mg, 0.64mmol). The mixture was stirred at room temperature for 16 h untilcompletion of the reaction was indicated by which TLC. The reactionsolution was then diluted with water (10 mL) and extracted with DCM(2×10 mL) then the combined organic layers were concentrated. Theresidue was purified by prep-HPLC to give the desired product (60 mg,Yield 25%). ¹H NMR (400 MHz, MeOD): 7.55 (s, 1H), 7.52 (s, 1H),7.24-7.15 (m, 3H), 6.85-6.73 (m, 4H), 6.03 (s, 1H), 4.22 (br.s, 1H),4.03-3.99 (m, 1H), 3.45 (s, 3H), 3.17-2.73 (m, 7H).LCMS (m/z): 391.2(M+1).

Compound 3(S)—N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)benzamide

Step 1: (R)-2-(oxiran-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline

To a solution of 1,2,3,4-tetrahydroisoquinoline (1 g, 7.52 mmol) in MeOH(40 mL) was added K₂CO₃ (5.19 g, 37.6 mmol) under 0° C. After stirringfor 30 minutes, (R)-2-(chloromethyl)oxirane (0.692 g, 7.52 mmol) wasadded the reaction. The mixture was then stirred at 0° C. overnightbefore filtration and washing of the solid by with MeOH. The solutionwas concentrated and the residue purified by column separation to givethe title compound as a colorless oil (70% purity). This crude was useddirectly in the next step. LCMS (m/z): 190.1 (M+1).

Step 2: (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol

To a solution of(R)-2-(oxiran-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline (200 mg, 5.2mmol) in EtOH (20 mL) was added NH₄OH (600 mg, 35.2 mmol) at −78° C. Thereaction mixture was then warmed and heated at 100° C. for 3 h in a sealtube. The reaction mixture was concentrated and the crude product wasused in next step without further purification. LCMS (m/z): 207.1 (M+1).

Step 3:(S)—N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)benzamide

A solution of (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol(200 mg, 0.97 mmol), benzoic acid (122.5 mg, 1.07 mmol), HATU (387.6 mg,1.02 mmol) and TEA (196.1 mg, 1.94 mmol) in DCM (20 mL) was stirred atroom temperature for 2 h until completion of the reaction. The reactionmixture was then diluted with water and extracted with DCM (20 ml×2).The combined organic layers were dried and concentrated with the residuepurified by pre-HPLC and SFC separation to give the desired compound (55mg, Yield 18%). ¹H NMR (400 MHz, MeOD): 7.66 (d, J=8.0 Hz, 2H),7.36-7.34 (m, 1H), 7.26 (d, J=7.6 Hz, 2H), 6.99-6.89 (m, 4H), 4.01-3.96(m, 1H), 3.61 (s, 2H), 3.43-3.37 (m, 2H), 2.77-2.72 (m, 4H), 2.56-2.53(m, 2H). LCMS (m/z): 311.1 (M+1).

Compound 8N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(pyridin-3-yl)benzamide

Step 1: methyl 3-(pyridin-3-yl)benzoate

A mixture of (3-(methoxycarbonyl)phenyl)boronic acid (600 mg, 3.33mmol), 3-bromopyridine (479 mg, 3.0 mmol), K₂CO₃ (1.2 g, 9.0 mmol) andPd(dppf)Cl₂ (50 mg) in a solution of dioxane (10 mL) and H₂O (2.5 mL)was stirred at 120° C. for 30 minutes with microwave heating under N₂.The catalyst was then filtered and the filtrate concentrated. Theresidue was then purified by column chromatography to give the desiredproduct and used directly in the next step. (630 mg Yield 90%).

Step 2: 3-(pyridin-3-yl)benzoic acid

To a solution of methyl 3-(pyridin-3-yl)benzoate (450 mg, 2.1 mmol) inMeOH (5 mL) was added aqueous of NaOH (1.5 mL, 0.4M). The mixture wasstirred at room temperature for 2 h then reaction solution wasconcentrated and the resulting residue dissolved in water and adjustedpH to 5-6 with 2N HCl. Extracted was then performed using EtOAc with theorganic layer dried and concentrated to give the target product whichwas used without further purification (600 mg, Yield 90%). LCMS (m/z):200.1 (M+1).

Step 3:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(pyridin-3-yl)benzamide

To a solution of 3-(pyridin-3-yl)benzoic acid (150 mg, 0.75 mmol) in DCM(6 mL) was added EDCI (215 mg, 1.10 mmol), HOBt (148 mg, 1.10 mmol),Et₃N (228 mg, 2.25 mmol) and1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (185 mg, 0.90mmol). The mixture was stirred at room temperature for 16 h. Thereaction solution was then washed with water and extracted with DCM. Theorganic layer was concentrated, dried and the residue purified byprep-HPLC to give the desired title product (110 mg, Yield 34%). ¹H NMR(400 MHz, MeOD) δ 8.80 (d, J=2.0 Hz, 1H), 8.52 (dd, J₁=4.8 Hz, J₂=3.6Hz, 1H), 8.10 (s, 1H), 8.09 (dd, J₁=8.8 Hz, J₂=1.6 Hz, 1H), 7.83 (d,J=7.6 Hz, 1H), 7.77 (d, J=7.6 Hz, 1H), 7.51-7.46 (m, 2H), 7.06-6.95 (m,4H), 4.15-4.10 (m, 1H), 3.69 (s, 2H), 3.60-3.47 (m, 2H), 2.85-2.79 (m,4H), 2.69-2.59 (m, 2H). LCMS (m/z): 388.2 (M+1).

Compound 9N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(pyridin-4-yl)benzamide

Step 1: methyl 3-(pyridin-4-yl)benzoate

A mixture of (3-(methoxycarbonyl)phenyl)boronic acid (600 mg, 3.33mmol), 4-bromopyridine (583.5 mg, 3.0 mmol), K₂CO₃ (1.2 g, 9.0 mmol) andPd(dppf)Cl₂ (50 mg) in a solution of dioxane (10 mL) and H₂O (2.5 mL)was stirred at 120° C. for 30 min with microwave heating. The catalystwas filtered and the filtrate concentrated. The residue was thenpurified by column chromatography to give the title product (630 mgYield 90%).

Step 2: 3-(pyridin-4-yl)benzoic acid

To a solution of methyl 3-(pyridin-4-yl)benzoate (450 mg, 2.1 mmol) inMeOH (5 mL) was added an aqueous solution of NaOH (1.5 mL, 0.4M). Themixture was stirred at room temperature for 2 h. The reaction solutionwas then concentrated, the residue was next dissolved in water andadjusted pH to 5-6 with the 2N HCl. After extraction with EtOAc, theorganic layers were dried and concentrated to give the product desired(600 mg, Yield 90%). LCMS (m/z): 200.1 (M+1).

Step 3:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(pyridin-4-yl)benzamide

To a solution of 3-(pyridin-4-yl)benzoic acid (300 mg, 1.5 mmol) in DCM(6 mL) was added EDCI (430 mg, 2.20 mmol), HOBt (296 mg, 2.20 mmol),Et₃N (556 mg, 4.50 mmol) and1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (370 mg, 1.80mmol). The mixture was stirred at room temperature for 16 h, then thereaction mixture was washed with water and extracted with DCM. Theorganic layer was then dried, concentrated and the residue purified byprep-HPLC to give the title product (230 mg, Yield 40%). ¹H NMR (400MHz, MeOD) δ 8.54 (d, J=4.0 Hz, 2H), 8.16 (s, 1H), 7.85-7.80 (m, 2H),7.64 (dd J=4.0 Hz, 2H), 7.48 (dd, J=7.6 Hz, 1H), 7.03-6.95 (m, 4H), 4.13(br.s, 1H), 3.66 (s, 2H), 3.60-3.48 (m, 2H), 2.80-2.77 (m, 4H),2.63-2.59 (m, 2H). LCMS (m/z): 388.2 (M+1).

Compound 11 (R)-phenyl(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamate

Step 1: (S)-2-(oxiran-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline

To a solution of 1,2,3,4-tetrahydroisoquinoline (5 g, 7.52 mmol) in THF(100 mL) was added KF (8.57 g, 150.4 mmol) at 0° C.(R)-oxiran-2-ylmethyl 3-nitrobenzenesulfonate (10.7 g, 41.4 mmol) wasadded to the reaction in 1 h. The solution was stirred at roomtemperature overnight. The solid was removed by filtration and washedwith THF. The solution was then concentrated and the residue used fornext step without further purification (11.3 g Yield 80%). LCMS (m/z):190.1 (M+1).

Step 2: (R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol

To a solution of(S)-2-(oxiran-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline (2.2 g, 0.012mol) in EtOH (30 mL), NH₃ was bubbled to the solution under −78° C. Thereaction mixture was then sealed and heated at 80° C. for 3 h. AfterLCMS indicated completion of the reaction, the mixture was concentratedand the crude product was used in next step without further purification(2.2 g, Yield 90%). LCMS (m/z): 207.1 (M+1).

Step 3: (R)-phenyl(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamate

To the stirring solution of(R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (200 mg, 0.97mmol) in 15 mL dry DCM was added TEA (1 mL) and the solution was cooledto 0° C. Phenyl carbonochloridate (151.3 mg, 1.02 mmol) in DCM (10 mL)was then added drop wise to the reaction over 20 minutes and thesolution was then stirred at room temperature overnight. The solutionwas then diluted with water, extracted with DCM, the organic layer wasconcentrated, purified by pre-HPLC to give the product as formate salt(125 mg, Yield 40%). ¹H NMR (400 MHz, MeOD) δ 7.35 (dd, J=7.6 Hz, 2H),7.31-7.18 (m, 5H), 7.08 (d, J=7.6 Hz, 2H), 4.33 (s, 2H), 4.22-4.19 (m,1H), 3.48 (t, J=6.0 Hz, 2H), 3.27-3.10 (m, 6H). LCMS (m/z): 327.2 (M+1).

Compound 12N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-(pyridin-2-yl)benzamide

Step 1: 2-(pyridin-2-yl)benzoic acid

A mixture of 2-boronobenzoic acid (400 mg, 2.4 mmol), 2-bromopyridine(416 mg, 2.6 mmol), K₂CO₃ (994 mg, 7.2 mmol) and Pd(dppf)Cl₂ (20 mg) indioxane (8 mL) and H₂O (2 mL) was stirred at 125° C. for 30 min. undermicrowave heating under N₂. The catalyst was filtered, and the filtratewas acidified with 2N HCl to pH 5-6. The solution was concentrated, andthe residue was dissolved in MeOH and filtered. The filtrate wasconcentrated, and the residue was purified by prep-TLC to give the titlecompound (205 mg, Yield 42.9%). LCMS (m/z): 200.0 (M+1).

Step 2:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-(pyridin-2-yl)benzamide

To a solution of 2-(pyridin-2-yl)benzoic acid (150 mg, 0.75 mmol) in DCM(6 mL) was added EDCI (215 mg, 1.1 mmol), HOBt (148 mg, 1.1 mmol), Et3N(228 mg, 2.25 mmol) and1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (185 mg, 0.9mmol). The mixture was stirred at 25° C. for 16 h. The reaction solutionwas washed with water and extracted with DCM. The organic layer was thenconcentrated, and the residue was purified by prep-HPLC to give thetitle compound (80 mg, Yield 27.5%). ¹H NMR (CD₃OD, 400 MHz): δ8.60-8.53 (m, 1H), 7.89-7.81 (m, 1H), 7.63-7.51 (m, 4H), 7.48-7.43 (m,1H), 7.39-7.32 (m, 1H), 7.12-7.05 (m, 3H), 7.05-6.98 (m, 1H), 4.05-3.93(m, 1H), 3.73-3.63 (s, 2H), 3.46-3.37 (m, 1H), 3.31-3.23 (m, 1H),2.92-2.75 (m, 4H), 2.56 (s, 2H). LCMS (m/z): 388.2 (M+1).

Compound 13N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4-(pyridin-2-yl)benzamide

Step 1: 4-(pyridin-2-yl)benzoic acid

A mixture of 4-boronobenzoic acid (200 mg, 1.2 mmol), 2-bromopyridine(208 mg, 1.3 mmol), K₂CO₃ (497 mg, 3.6 mmol) and Pd(dppf)Cl₂ (10 mg) indioxane (4 mL) and H₂O (1 mL) was stirred at 125° C. for 30 min withmicrowave heating under N₂. The catalyst was filtered, and the filtratewas acidified with 2N HCl to pH 5-6. The solution was concentrated, andthe residue was dissolved in MeOH and filtered. The filtrate wasconcentrated, and the residue was purified by prep-TLC to give the titlecompound (100 mg, Yield 41.8%). LCMS (m/z): 200.1 (M+1).

Step 2:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4-(pyridin-2-yl)benzamide

To a solution of 4-(pyridin-2-yl)benzoic acid (100 mg, 0.5 mmol) in DCM(5 mL) was added EDCI (144 mg, 0.75 mmol), HOBt (101 mg, 0.75 mmol),Et₃N (152 mg, 1.5 mmol) and1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (103 mg, 0.5mmol). The mixture was stirred at 25° C. for 16 h. The reaction solutionwas washed with water and extracted with DCM. The organic layer wasconcentrated, and the residue was purified by prep-HPLC to give thetitle compound (30 mg, Yield 15.5%). ¹H NMR (CD₃OD, 400 MHz): δ8.70-8.60 (m, 1H), 8.01-7.84 (m, 6H), 7.45-7.36 (m, 1H), 7.16-6.99 (m,4H), 4.20-4.10 (m, 1H), 3.79 (s, 2H), 3.62-3.46 (m, 2H), 2.92 (s, 4H),2.78-2.65 (m, 2H). LCMS (m/z): 388.2 (M+1).

Compound 14N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-morpholinobenzamide

Step 1:3-bromo-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)benzamide

To a solution of 3-bromobenzoic acid (200 mg, 1.0 mmol) in DCM (8 mL)was added Et₃N (303 mg, 3.0 mmol), EDCI (383 mg, 2.0 mmol), HOBt (270mg, 2.0 mmol) and 1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol(247 mg, 1.2 mmol). The mixture was stirred at 25° C. for 6 h. Themixture was treated with water and extracted with EA. The organic layerwas washed with NaHCO₃, brine, dried over Na₂SO₄ and concentrated togive the title compound which was used in next step without furtherpurification (300 mg, Yield 77%). LCMS (m/z): 390.1 (M+1).

Step 2:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-morpholinobenzamide

A mixture of3-bromo-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)benzamide(200 mg, 0.51 mmol), morpholine (44 mg, 0.51 mmol), Pd₂(dba)₃ (46 mg,0.05 mmol), BINAP (62 mg, 0.1 mmol) and NaOtBu (73 mg, 0.77 mmol) intoluene (6 mL) was stirred at reflux for 16 h under N₂. The reactionsolution was concentrated, and the residue was dissolved in EA andfiltered. The filtrate was concentrated, and the residue was purified byprep-HPLC to give the title compound (15 mg, Yield 7.5%). ¹H NMR (CD₃OD,400 MHz): δ 8.48 (brs, 1H), 7.42 (s, 1H), 7.37-7.28 (m, 2H), 7.27-7.19(m, 3H), 7.18-7.11 (m, 2H), 4.31-4.23 (m, 1H), 4.19 (s, 2H), 3.86 (dd,J=5.1, 4.8 Hz, 4H), 3.61-3.44 (m, 2H), 3.32-3.29 (m, 2H), 3.25-3.16 (m,4H), 3.14-2.97 (m, 4H). LCMS (m/z): 396.2 (M+1).

Compound 15N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(((tetrahydro-2H-pyran-4-yl)amino)methyl)benzamide

Step 1: methyl 3-(((tetrahydro-2H-pyran-4-yl)amino)methyl)benzoate

To a solution of methyl 3-formylbenzoate (492 mg, 3.0 mmol) in MeOH (10mL) was added tetrahydro-2H-pyran-4-amine (303 mg, 3.0 mmol) and AcOH(0.05 mL). The mixture was stirred at 25° C. for 2 h. NaBH₃CN (945 mg,15.0 mmol) was added, and the resulting mixture was stirred at 25° C.for 16 h. The reaction solution was concentrated and the residue wasdissolved in water and extracted with DCM. The organic layer wasconcentrated, and the residue was purified by prep-TLC to give the titleproduct (500 mg, Yield 67%). LCMS (m/z): 250.1 (M+1).

Step 2: methyl3-(((tert-butoxycarbonyl)(tetrahydro-2H-pyran-4-yl)amino)-methyl)benzoate

To a solution of methyl3-(((tetrahydro-2H-pyran-4-yl)amino)methyl)benzoate (400 mg, 1.6 mmol)in a mixture solution of THF (10 mL) and H₂O (1 mL) was added Boc₂O (418mg, 1.9 mmol) and Et₃N (243 mg, 2.4 mmol). The mixture was stirred at25° C. for 16 h. The reaction solution was concentrated to remove THF,and the residue was dissolved in water and extracted with EA. Theorganic layer was concentrated, and the residue was purified by columnchromatography to give the title product (550 mg, 98%). LCMS (m/z):350.1 (M+1).

Step 3:3-(((tert-butoxycarbonyl)(tetrahydro-2H-pyran-4-yl)amino)methyl)benzoicacid

To a solution of methyl3-(((tert-butoxycarbonyl)(tetrahydro-2H-pyran-4-yl)amino)methyl)benzoate(550 mg, 1.57 mmol) in MeOH (5 mL) was added aqueous of NaOH (2.0 mL,40% w/w). The mixture was stirred at 25° C. for 4 h. The reactionsolution was concentrated, and the residue was dissolved in water andadjusted pH to 5-6 with 2N of HCl and extracted with EA. The organiclayer was concentrated to give the desired product (300 mg, Yield 57%).LCMS (m/z): 336.1 (M+1).

Step 4: tert-butyl3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)benzyl(tetrahydro-2H-pyran-4-yl)carbamate

To a solution of3-(((tert-butoxycarbonyl)(tetrahydro-2H-pyran-4-yl)amino)methyl)benzoicacid (300 mg, 0.89 mmol) in DCM (8 mL) was added EDCI (257 mg, 1.34mmol), HOBt (181 mg, 1.34 mmol), Et₃N (270 mg, 2.67 mmol) and1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (183 mg, 0.89mmol). The mixture was stirred at 25° C. for 16 h. The reaction solutionwas washed with water and extracted with DCM. The organic layer wasconcentrated to give the title product (350 mg, Yield 65%). LCMS (m/z):524.3 (M+1).

Step 5:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(((tetrahydro-2H-pyran-4-yl)amino)methyl)benzamide

To a solution of tert-butyl3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)benzyl(tetrahydro-2H-pyran-4-yl)carbamate(450 mg, crude) in DCM (6 mL) was added TFA (6 mL). The mixture wasstirred at 25° C. for 16 h. The reaction solution was concentrated, andthe residue was purified by prep-HPLC to give the title product (200 mg,54.9%). ¹H NMR (CD₃OD, 400 MHz): δ 8.08 (s, 1H), 8.00-7.89 (m, 1H),7.81-7.68 (m, 1H), 7.58 (s, 1H), 7.39-7.15 (m, 4H), 4.75-4.47 (m, 2H),4.46-4.39 (m, 1H), 4.34 (s, 2H), 4.05 (dd, J=11.6, 3.6 Hz, 2H),3.98-3.70 (brs, 1H), 3.62-3.55 (m, 2H), 3.55-3.45 (m, 4H), 3.45-3.32 (m,2H), 3.32-3.06 (m, 2H), 2.22-2.07 (m, 2H), 1.89-1.72 (m, 2H). LCMS(m/z): 424.2 (M+1).

Compound 16N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-((tetrahydro-2H-pyran-4-yl)amino)benzamide

Step 1: 3-((tert-butoxycarbonyl)amino)benzoic acid

To a solution of 3-aminobenzoic acid (1.37 g, 10 mmol) in a mixturesolution of THF (20 mL) and H₂O (2 mL) was added Boc₂O (2.18 g, 10 mmol)and Et₃N (1.52 g, 15 mmol). The mixture was stirred at 25° C. for 16 h.The reaction solution was concentrated and the residue was dissolved inwater and extracted with EA. The organic layer was concentrated to givethe title product (2.3 g, Yield 97%). LCMS (m/z): 260.0 (M+23).

Step 2: tert-butyl(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)phenyl)carbamate

To a solution of 3-((tert-butoxycarbonyl)amino)benzoic acid (2.5 g, 10.5mmol) in DCM (25 mL) was added EDCI (3.0 g, 15.7 mmol), HOBt (2.1 g,15.7 mmol), Et₃N (2.1 g, 21 mmol) and1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (2.2 g, 10.5mmol). The mixture was stirred at 25° C. for 16 h. The reaction solutionwas washed with water, extracted with DCM and the organic layer wasconcentrated, and the residue was purified by column chromatography togive the desired product (3.2 g, Yield 71%). LCMS (m/z): 426.3 (M+1).

Step 3:3-amino-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)benzamide

To a solution of tert-butyl(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)phenyl)carbamate(500 mg, 1.18 mmol) in DCM (5 mL) was added TFA (5 mL). The mixture wasstirred at 25° C. for 16 h. The reaction solution was concentrated, andthe residue was dissolved in water, pH was adjusted to 7˜7.5 withsaturated aqueous NaHCO₃ and extracted with EA. The organic layer wasconcentrated to give the title product (360 mg, Yield 94%). The crudeproduct was used in next step without further purification. LCMS (m/z):326.2 (M+1).

Step 4:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-((tetrahydro-2H-pyran-4-yl)amino)benzamide

To a solution of3-amino-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)benzamide(325 mg, 1.0 mmol) in MeOH (10 mL) was added dihydro-2H-pyran-4(3H)-one(88 mg, 1.0 mmol) and AcOH (0.05 mL). The mixture was stirred at 25° C.for 2 h. NaBH₃CN (630 mg, 10.0 mmol) was added, and the resultingmixture was stirred at 25° C. for 16 h. The reaction solution wasconcentrated, and the residue was dissolved in water, extracted with EA.The organic layer was concentrated, and the residue was purified byprep-HPLC to give the title compound (200 mg, Yield 48.9%). ¹H NMR(CD₃OD, 400 MHz): δ 8.44 (brs, 1H), 7.32-7.20 (m, 3H), 7.20-7.13 (m,2H), 7.13-7.09 (m, 1H), 7.08-7.00 (m, 1H), 6.86-6.77 (m, 1H), 4.39 (s,2H), 4.35-4.25 (m, 1H), 4.03-3.89 (m, 2H), 3.63-3.40 (m, 7H), 3.31-3.07(m, 4H), 2.06-1.92 (m, 2H), 1.55-1.40 (m, 2H). LCMS (m/z): 410.2 (M+1).

Compound 17N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)picolinamide

Step 1:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)picolinamide

To a solution of picolinic acid (100 mg, 0.81 mmol) in DCM (10 mL), wasadded EDCI (187 mg, 0.97 mmol) and HOBT (132 mg, 0.98 mmol), which wasstirred at 25° C. for 0.5 h before1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (167 mg, 0.81mmol) was added and the resulting mixture was stirred at 25° C. for 2 h.The solution was concentrated in vacuo and the residue was purified byprep-HPLC to provide the title compound (68 mg, Yield 26.9%). ¹H NMR(CD₃OD, 400 MHz): δ 8.61 (d, J=3.9 Hz, 1H), 8.49 (brs, 1H), 8.10 (d,J=7.8 Hz, 1H), 8.01-7.92 (m, 1H), 7.56 (dd, J=5.1, 6.8 Hz, 1H),7.31-7.20 (m, 3H), 7.19-7.13 (m, 1H), 4.44-4.27 (m, 3H), 3.66-3.47 (m,4H), 3.31-3.12 (m, 4H). LCMS (m/z): 312.1 (M+1).

Compound 21N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4-sulfamoylbenzamide

Step 1:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4-sulfamoylbenzamide

A solution of 4-sulfamoylbenzoic acid (88.4 mg, 0.44 mmol), HATU (182.4mg, 0.48 mmol) and TEA (48.48 mg, 0.48 mmol) in DCM (10 mL) was stirredat 22° C. for 10 min.1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (100 mg, 0.48mmol) was then added and the solution was stirred at 22° C. for another3 h. The reaction mixture was diluted with water and extracted with DCM.The organic layers were combined, dried over anhydrous Na₂SO₄, filteredand concentrated. The residue was purified by prep-HPLC to give thetitle compound (49.5 mg, Yield 29%). ¹H NMR (CD₃OD, 400 MHz): δ 7.92 (s,4H), 7.16-7.09 (m, 3H), 7.05-7.02 (m, 1H), 4.14-4.12 (m, 1H), 3.77 (s,2H), 3.58-3.39 (m, 2H), 2.94-2.91 (m, 2H), 2.90-2.86 (m, 2H), 2.75-2.66(m, 2H). LCMS (m/z): 390.1 (M+1).

Compound 234-acetamido-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)benzamide

Step 1:4-acetamido-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)benzamide

A solution of 4-acetamidobenzoic acid (100 mg, 0.56 mmol), HATU (234 mg,0.62 mmol) and TEA (63 mg, 0.62 mmol) in DCM (10 mL) was stirred at 22°C. for 10 min. 1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol(116 mg, 0.56 mmol) was then added and the solution was stirred at 22°C. for another 3 h. The reaction mixture was then diluted with water andextracted with DCM. The organic layers were combined and dried overanhydrous Na₂SO₄, filtered and concentrated and the residue was purifiedby prep-HPLC to give the title compound (48.5 mg, Yield 24%). ¹H NMR(CD₃OD, 400 MHz): δ 7.77-7.72 (m, 2H), 7.63-7.57 (m, 2H), 7.17-7.08 (m,3H), 7.04 (d, J=7.0 Hz, 1H), 4.12 (t, J=6.0 Hz, 1H), 3.75 (s, 2H),3.58-3.46 (m, 2H), 2.92-2.85 (m, 4H), 2.74-2.63 (m, 2H), 2.16 (s, 3H).LCMS (m/z): 368.1 (M+1).

Compound 28N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(2-(dimethylamino)ethoxy)benzamide

Step 1: methyl 3-(2-(dimethylamino)ethoxy)benzoate

To a stirred mixture of methyl 3-hydroxybenzoate (200 mg, 1.32 mmol),and K₂CO₃ (169 mg, 1.58 mmol) in MeCN (50 mL) was added2-chloro-N,N-dimethylethanamine (137 mg, 1.58 mmol). The mixture wasstirred at 60° C. for 16 h. The reaction mixture was filtered and thefiltrate was concentrated to give the title compound that was usedwithout further purification (300 mg, Yield 98%). ¹H NMR (CDCl₃, 400MHz): δ 7.61-7.53 (m, 1H), 7.53-7.47 (m, 1H), 7.26-7.23 (m, 1H),7.06-7.04 (m, 1H), 4.05 (t, J=5.6 Hz, 2H), 3.84 (s, 3H), 2.69 (t, J=5.6Hz, 2H), 2.28 (s, 6H). LCMS (m/z): 224.2 (M+1).

Step 2:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(2-(dimethylamino)ethoxy)benzamide

A mixture of crude methyl 3-(2-(dimethylamino)ethoxy)benzoate (300 mg,1.34 mmol) and 1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol(332 mg, 1.61 mmol) in EtOH (2 mL) was heated at 120° C. in a microwavereactor for 3 h. After evaporation of the solvent, the residue waspurified by prep-HPLC to give the title compound (34 mg, Yield 6.4%). ¹HNMR (CD₃OD, 400 MHz): δ 7.43-7.42 (m, 1H), 7.34-7.29 (m, 2H), 7.12-7.10(m, 4H), 7.09-7.03 (m, 1H), 4.20-4.10 (m, 3H), 3.75 (brs, 2H), 3.59-3.42(m, 2H), 2.95-2.85 (m, 4H), 2.82-2.77 (m, 2H), 2.72-2.65 (m, 2H), 2.37(s, 6H). LCMS (m/z): 398.1 (M+1).

Compound 30N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-((4-methylpiperazin-1-yl)methyl)benzamide

Step 1: 3-((4-(tert-butoxycarbonyl)piperazin-1-yl)methyl)benzoic acid

The solution of 3-formylbenzoic acid (300 mg, 1.83 mmol) and tert-butylpiperazine-1-carboxylate (340 mg, 1.83 mmol) in MeOH (10 mL) was stirredat 27° C. for 1 h. Then NaBH₃CN (138 mg, 2.19 mmol) was added to thesolution and stirred at 27° C. for 6 h. The solution was concentratedand the residue was purified by column to give the title product (320mg, Yield 50%). LCMS (m/z): 321.2 (M+1).

Step 2: tert-butyl4-(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)benzyl)piperazine-1-carboxylate

The solution of 3-((4-(tert-butoxycarbonyl)piperazin-1-yl)methyl)benzoicacid (100 mg, 0.31 mmol) and HATU (119 mg, 0.31 mmol) in DCM (10 mL) wasstirred at 28° C. for 30 min. Then1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (64.4 mg, 0.31mmol) and DIPEA (48.4 mg, 0.38 mmol) was added and the resultingsolution was stirred at 28° C. for 16 h. The solution was concentratedand the residue was purified by column chromatography to give the crudetitle product (150 mg, Yield 94%). LCMS (m/z): 509.2 (M+1).

Step 3:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(piperazin-1-ylmethyl)benzamide

The solution of tert-butyl4-(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)benzyl)piperazine-1-carboxylate(160 mg, 0.314 mmol) in DCM (2 mL) and TFA (2 mL) was stirred at 27° C.for 16 h. The solution was concentrated and the residue was purified byprep-HPLC to give the title product (89 mg, Yield 69.0%). ¹H NMR (D₂O,400 MHz): δ 7.66-7.56 (m, 2H), 7.51-7.44 (m, 1H), 7.44-7.37 (m, 1H),7.16-7.06 (m, 3H), 7.02 (d, J=7.3 Hz, 1H), 4.11 (quin, J=5.9 Hz, 1H),3.73-3.60 (m, 2H), 3.56-3.49 (m, 2H), 3.49-3.42 (m, 1H), 3.41-3.32 (m,1H), 2.86-2.75 (m, 8H), 2.68-2.58 (m, 2H), 2.56-2.32 (m, 4H). LCMS(m/z): 409.2 (M+1).

Step 4:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(4-methylpiperazin-1-yl)methyl)benzamide

The solution ofN-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(piperazin-1-ylmethyl)benzamide(78 mg, 0.19 mmol) and HCHO solution (0.5 mL) in MeOH (10 mL) wasstirred at 27° C. for 1 h. Then NaBH₃CN (14.5 mg, 0.23 mmol) was addedto the solution and stirred at 27° C. for 4 h. The solution wasconcentrated and the residue was purified by column chromatography togive the title product (14.1 mg, Yield 17.5%). ¹H NMR (CD₃OD, 400 MHz):δ 7.79 (s, 1H), 7.70 (d, J=7.8 Hz, 1H), 7.50 (d, J=7.5 Hz, 1H),7.39-7.32 (m, 1H), 7.20-7.07 (m, 3H), 7.06-6.98 (m, 1H), 4.13 (quin,J=6.0 Hz, 1H), 3.75 (s, 2H), 3.63-3.44 (m, 4H), 2.95-2.83 (m, 4H),2.78-2.62 (m, 3H), 2.62-2.30 (m, 7H), 2.28 (s, 3H). LCMS (m/z): 423.2(M+1).

Compound 34N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(1-methylpyrrolidin-2-yl)benzamide

To a solution ofN-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(pyrrolidin-2-yl)benzamide(20 mg, 0.13 mmol) in MeOH (20 mL) was added HCHO (1 mL) and AcOH (0.05mL). The reaction mixture was stirred at room temperature for 30 min atwhich time NaBH₃CN (200 mg, 3.22 mmol) was added. The reaction mixturewas stirred at room temperature for 2 h. The solvent was removed and thecrude product was purified by prep-HPLC to give the desired product (8.5mg, Yield 16.8%). ¹H NMR (CD₃OD, 400 MHz): δ 7.80 (brs, 1H), 7.67 (d,J=7.6 Hz, 1H), 7.52 (d, J=7.6 Hz, 1H), 7.35-7.41 (m, 1H), 7.09-7.15 (m,3H), 7.09-7.15 (m, 1H), 7.02-7.08 (m, 1H), 4.10-4.16 (m, 1H), 3.73-3.81(m, 2H), 3.49-3.58 (m, 2H), 3.20-3.28 (m, 1H), 3.08-3.16 (m, 1H),2.84-2.97 (m, 4H), 2.64-2.75 (m, 2H), 2.33-2.40 (m, 1H), 2.20-2.27 (m,1H), 2.16 (s, 3H), 1.95-2.05 (m, 1H), 1.77-1.93 (m, 2H). LCMS (m/z):394.1 (M+1).

Compound 35N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(piperazin-1-yl)benzamide

Step 1: 3-(4-(tert-butoxycarbonyl)piperazin-1-yl)benzoic acid

The mixture of ethyl 3-bromobenzoate (500 mg, 2.33 mmol), tert-butylpiperazine-1-carboxylate (433 mg, 2.33 mmol) and NaOtBu (268 mg, 2.78mmol), Pd₂(dba)₃ (20 mg, 0.034 mmol) and Xantphos (20 mg, 0.034 mmol) inanhydrous dioxane (10 mL) was heated to 110° C. for 10 h. The mixturewas concentrated and the residue was partitioned in water, the solutionwas adjusted to pH=5, and extracted with DCM. The organic layer waswashed with water and brine, dried over anhydrous Na₂SO₄, filtered andconcentrated to afford the title compound that was used for next step(300 mg, Yield 42.2%). LCMS (m/z): 307.1 (M+1).

Step 2: tert-butyl4-(3-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropylcarbamoyl)phenyl)piperazine-1-carboxylate

The solution of 3-(4-(tert-butoxycarbonyl)piperazin-1-yl)benzoic acid(300 mg, 1.0 mmol) and HATU (381 mg, 1.0 mmol) in DCM (10 mL) wasstirred at 25° C. for 30 min. Then1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (200 mg, 1.0mmol) and DIPEA (259 mg, 2.00 mmol) was added and the resulting solutionwas stirred at 25° C. for 16 h. The solution was concentrated and theresidue was purified by column chromatography to give the title product(140 mg, Yield 28.8%). LCMS (m/z): 495.2 (M+1).

Step 3:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(piperazin-1-yl)benzamide

To a solution of tert-butyl4-(3-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropylcarbamoyl)phenyl)piperazine-1-carboxylate (140 mg, 0.28 mmol) inDCM (2 mL) was added TFA (2 mL). The resulting solution was stirred at27° C. for 4 h. The solution was concentrated and the residue waspurified by prep-HPLC to give the title product (64.0 mg, Yield 57%). ¹HNMR (CD₃OD, 400 MHz): δ 7.47-7.38 (s, 1H), 7.31-7.21 (m, 2H), 7.19-7.08(m, 4H), 7.08-7.01 (m, 1H), 4.13 (quin, J=6.0 Hz, 1H), 3.77 (s, 2H),3.62-3.52 (m, 1H), 3.51-3.43 (m, 1H), 3.31-3.19 (m, 4H), 3.15-3.00 (m,4H), 2.98-2.83 (m, 4H), 2.75-2.62 (m, 2H). LCMS (m/z): 395.2 (M+1).

Compound 38N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(1-methylpyrrolidin-3-yl)benzamide

Step 1: tert-butyl3-(((trifluoromethyl)sulfonyl)oxy)-2,5-dihydro-1H-pyrrole-1-carboxylate

A solution of tert-butyl 3-oxopyrrolidine-1-carboxylate (5 g, 27.0 mmol)in THF (50 ml) was slowly added to a stirring solution of NaHMDS (1MTHF, 32.4 ml, 32.4 mmol) at −78° C. After 10 min a solution ofN-phenyl-O-((trifluoromethyl)sulfonyl)-N-(((trifluoromethyl)sulfonyl)oxy)hydroxylamine (10.6 g, 29.7 mmol) in THF (50 ml) was slowlyadded. Stirring at −78° C. was continued for 30 min and the cooling bathwas removed. The reaction mixture was stirred at room temperature for1.5 h. The mixture was cooled to 0° C., quenched with sat. NaHCO₃, andextracted with MTBE. The organic layer was washed with 5% citric acid,1M NaOH, H₂O, brine, dried over Na₂SO₄, concentrated and the residue waspurified by flash column chromatography to give the title compound (1.5g, Yield 17.4%). ¹H NMR (CDCl₃, 400 MHz): δ 5.77 (s, 1H), 4.14-4.30 (m,4H), 1.48 (s, 9H).

Step 2: tert-butyl3-(3-(methoxycarbonyl)phenyl)-2,5-dihydro-1H-pyrrole-1-carboxylate

To a solution of tert-butyl3-(((trifluoromethyl)sulfonyl)oxy)-2,5-dihydro-1H-pyrrole-1-carboxylate(300 mg, 0.95 mmol) in dioxane (4 mL) and H₂O (1 mL) was added methyl3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (298 mg, 1.13mmol), Pd(dppf)Cl₂ (66 mg, 0.09 mmol) and K₂CO₃ (392 mg, 2.84 mmol) at27° C. The mixture was stirred at 100° C. for 16 h. The catalyst wasfiltered, the filtrate was concentrated and the residue was purified bycolumn chromatography to give the title compound (213 mg, Yield 74.2%).¹H NMR (CDCl₃, 400 MHz): δ 8.03 (d, J=19.6 Hz, 1H), 7.94 (d, J=7.8 Hz,1H), 7.55 (dd, J=15.7, 7.8 Hz, 1H), 7.38-7.45 (m, 1H), 6.22 (dt, J=16.4,1.8 Hz, 1H), 4.43-4.58 (m, 2H), 4.24-4.38 (m, 2H), 3.88-3.96 (m, 3H),1.51 (d, J=7.9 Hz, 9H).

Step 3: tert-butyl3-(3-(methoxycarbonyl)phenyl)pyrrolidine-1-carboxylate

To a solution of tert-butyl3-(3-(methoxycarbonyl)phenyl)-2,5-dihydro-1H-pyrrole-1-carboxylate (213mg, 0.7 mmol) in MeOH (10 mL) was added Pd/C (20 mg). The mixture wasstirred for 30 min at 30° C. under H₂ atmosphere. The mixture wasfiltered and the filtrate was concentrated to give the title compoundwhich was used in next step without further purification (210 mg, Yield98.1%). ¹H NMR (CDCl₃, 400 MHz): δ 7.88-7.97 (m, 2H), 7.36-7.48 (m, 2H),3.92 (s, 3H), 3.77-3.90 (m, 1H), 3.53-3.72 (m, 1H), 3.25-3.47 (m, 3H),2.29 (d, J=5.27 Hz, 1H), 2.01 (quin, J=10.2 Hz, 1H), 1.42-1.55 (m, 10H).

Step 4: 3-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)benzoic acid

To a solution of tert-butyl3-(3-(methoxycarbonyl)phenyl)pyrrolidine-1-carboxylate (210 mg, 0.7mmol) in EtOH (4 ml) was added a solution of NaOH (56 mg, 1.4 mmol) inH₂O (1 ml) at 29° C. The mixture was stirred for 30 min at 29° C. Themixture was concentrated and the residue was treated with water andextracted with EA. The water layer was treated with 2N HCl until pH=3,extracted with EA and the combined organic layers were washed withbrine, dried over Na₂SO₄ and concentrated to give the title compoundwhich was used in next step without further purification (200 mg, Yield98.0%).

Step 5: tert-butyl3-(34(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)phenyl)pyrrolidine-1-carboxylate

To a solution of 3-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)benzoic acid(200 mg, 0.69 mmol) in DMF (4 ml) was added TEA (208 mg, 2.06 mmol),HOBt (139 mg, 1.03 mmol), EDCI (197 mg, 1.03 mmol) and1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (170 mg, 0.82mmol) at 33° C. The reaction mixture was stirred for 16 h at 31° C. Themixture was treated with water and extracted with EA. The organic layerwas washed with brine, dried over Na₂SO₄ and concentrated to give thetitle compound which was used in next step without further purification(300 mg, Yield 92%).

Step 6:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(pyrrolidin-3-yl)benzamide

To a solution of tert-butyl3-(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)phenyl)pyrrolidine-1-carboxylate(400 mg, 0.83 mmol) in CH₂Cl₂ (5 mL) was added TFA (1 mL) at 29° C. Themixture was stirred for 2 h at 29° C. The mixture was concentrated andthe residue was purified by prep-HPLC to give the title compound (79.1mg, Yield 25.0%). ¹H NMR (CD₃OD, 400 MHz): δ 7.75-7.93 (m, 2H),7.45-7.62 (m, 2H), 7.17-7.37 (m, 4H), 4.45-4.74 (m, 2H), 4.40 (dd,J=6.3, 3.3 Hz, 1H), 3.71-4.04 (m, 2H), 3.49-3.70 (m, 5H), 3.35-3.49 (m,3H), 3.08-3.32 (m, 3H), 2.52 (qd, J=6.6, 4.2 Hz, 1H), 2.09-2.27 (m, 1H).LCMS (m/z): 380.2 (M+1).

Step 7:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(1-methylpyrrolidin-3-yl)benzamide

To a solution ofN-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(pyrrolidin-3-yl)benzamide(200 mg, 0.53 mmol) in MeOH (4 ml) was added HCHO (31.9 mg, 1.05 mmol)and NaBH₃CN (66.1 mg, 1.05 mmol) at 29° C. The mixture was then addedAcOH (0.5 ml) at 29° C. and stirred for 16 h. The mixture was purifiedby prep-HPLC to give the title compound (29.6 mg, Yield 14.3%). ¹H NMR(CD₃OD, 400 MHz): δ 8.52 (brs, 2H), 7.88 (s, 1H), 7.78 (d, J=7.7 Hz,1H), 7.53-7.60 (m, 1H), 7.43-7.52 (m, 1H), 7.22-7.33 (m, 3H), 7.15-7.21(m, 1H), 4.36 (s, 3H), 3.78 (brs, 2H), 3.46-3.67 (m, 6H), 3.41 (brs,1H), 3.07-3.19 (m, 3H), 3.01 (s, 3H), 2.50-2.64 (m, 1H), 2.19-2.34 (m,1H). LCMS (m/z): 394.2 (M+1).

Compound 40N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-methyl-3-((tetrahydro-2H-pyran-4-yl)amino)benzamide

Step 1:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-methyl-3-nitrobenzamide

To a solution of 2-methyl-3-nitrobenzoic acid (1.0 g, 5.5 mmol) in DCM(20 mL) was added EDCI (1.58 g, 8.25 mmol), HOBt (1.11 g, 8.25 mmol),Et₃N (1.11 g, 11.0 mmol) and1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (1.36 g, 6.6mmol). The mixture was stirred at 25° C. for 16 h. The reaction solutionwas washed with water and extracted with DCM. The organic layer wasconcentrated, and the residue was purified by column chromatography togive the title product (1.6 g, 78.8%). LCMS (m/z): 370.2 (M+1).

Step 2:3-amino-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-methylbenzamide

To a solution ofN-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-methyl-3-nitrobenzamide(1.6 g, 4.3 mmol) in EtOH (15 mL) and H₂O (15 mL) was added Fe powder(1.45 g, 25.8 mmol) and NH₄Cl (1.38 g, 25.8 mmol). The mixture wasstirred at 60° C. for 4 h. The reaction solution was filtered, and thefiltrate was concentrated to remove EtOH. The residue was diluted withwater and extracted with DCM. The organic layer was concentrated to givethe desired product (1.4 g, Yield 95.9%). The crude product was used innext step without further purification. LCMS (m/z): 340.1 (M+H).

Step 3:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-methyl-3-((tetrahydro-2H-pyran-4-yl)amino)benzamide

To a solution of3-amino-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-methylbenzamide(200 mg, 0.59 mmol) in MeOH (8 mL) was added AcOH (0.05 mL) anddihydro-2H-pyran-4(3H)-one (118 mg, 1.18 mmol). The mixture was stirredat 25° C. for 2 h. NaBH₃CN (186 mg, 2.95 mmol) was added and theresulting mixture was stirred at 25° C. for 2 h. The reaction solutionwas concentrated and the residue was washed with water and extractedwith EA. The organic layer was concentrated, and the residue waspurified by prep-HPLC to give the title compound (24 mg, Yield 9.6%). ¹HNMR (CD₃OD, 400 MHz): δ 8.41 (s, 1H), 7.35-7.23 (m, 3H), 7.20 (d, J=7.0Hz, 1H), 7.11 (t, J=7.8 Hz, 1H), 6.81 (d, J=8.3 Hz, 1H), 6.70 (d, J=7.3Hz, 1H), 4.44 (s, 2H), 4.33 (brs, 1H), 3.99 (d, J=11.5 Hz, 2H),3.66-3.43 (m, 7H), 3.38-3.16 (m, 4H), 2.15 (s, 3H), 2.01 (d, J=12.8 Hz,2H), 1.63-1.48 (m, 2H). LCMS (m/z): 424.2 (M+1).

Compound 42N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(pyrrolidin-2-yl)benzamide

Step 1: tert-butyl2-(34(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)phenyl)pyrrolidine-1-carboxylate

A mixture of compound 3-(1-(tert-butoxycarbonyl)pyrrolidin-2-yl)benzoicacid (100 mg, 0.34 mmol),1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (70 mg, 0.34mmol), BOPCl (100 mg, 0.41 mmol) and DIPEA (1 mL) in DCM (10 mL) wasstirred at 25° C. for 4 h. The reaction mixture was diluted with waterand extracted with DCM. The combined organic layers were washed withbrine, dried over Na₂SO₄, concentrated and the residue was purified byprep-TLC to give the title product which was used directly in next step(150 mg, Yield 93%). LCMS (m/z): 480.2 (M+1).

Step 2:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(pyrrolidin-2-yl)benzamide

To a solution of tert-butyl2-(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)phenyl)pyrrolidine-1-carboxylate(100 mg, 0.11 mmol) in EA (10 mL) was added HCl (1M in EA, 4 mL). Thereaction mixture was stirred at 25° C. for 16 h. The solvent was thenremoved by in vacuo and the crude product was purified by prep-HPLC togive the title compound (39.4 mg, Yield 52%). ¹H NMR (CD₃OD, 400 MHz): δ7.80 (brs, 1H), 7.66 (d, J=7. Hz, 1H), 7.53 (d, J=7.6 Hz, 1H), 7.32-7.43(m, 1H), 7.00-7.17 (m, 4H), 4.05-4.24 (m, 2H), 3.73-3.81 (m, 2H),3.48-3.60 (m, 2H), 3.17-3.27 (m, 1H), 2.96-3.07 (m, 1H), 2.81-2.95 (m,4H), 2.64-2.75 (m, 2H), 2.20-2.32 (m, 1H), 1.87-2.05 (m, 2H), 1.70-1.84(m, 1H). LCMS (m/z): 380.2 (M+1).

Compound 44(S)—N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-((tetrahydro-2H-pyran-4-yl)amino)benzamide

Step 1: (R)-2-(oxiran-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline

To a solution of 1,2,3,4-tetrahydroisoquinoline (10 g, 0.15 mol) in THF(100 mL) at 0° C. was added KF (22 g, 0.3 mmol). After 1 h,(S)-oxiran-2-ylmethyl 3-nitrobenzenesulfonate (21.4 g, 0.17 mmol) wasadded and the resulting solution was stirred at 22° C. for 16 h. Thesolid was removed by filtration and washed with THF. The solution wasconcentrated and the crude compound was used for next step withoutfurther purification (15 g, Yield 53%). LCMS (m/z): 190.1 (M+1).

Step 2: (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol

To a solution of(R)-2-(oxiran-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline (15 g, 0.08mol) in EtOH (100 mL) at −78° C. was slowly bubbled NH₃ (g). Thereaction mixture was then sealed and heated at 80° C. for 3 h. Thereaction mixture was concentrated and the crude product was used in nextstep without further purification (15 g, Yield 92%). LCMS (m/z): 207.1(M+1).

Step 3: Methyl 3-((tert-butoxycarbonyl)amino)benzoate

To a solution of methyl 3-aminobenzoate (2.0 g, 13.2 mmol) in THF (20mL) was added Et₃N (2.67 g, 26.4 mmol) and Boc₂O (3.16 g, 14.5 mmol) at0° C. The mixture was stirred at 25° C. for 16 h. The reaction solutionwas concentrated to remove THF, and the residue was washed with waterand extracted with EA. The organic layer was concentrated, and theresidue was purified by column chromatography to give the title product(1.6 g, Yield 48.5%). ¹H NMR (CD₃OD, 400 MHz): δ 8.12 (s, 1H), 7.64-7.60(m, 2H), 7.37-7.33 (t, J=8 Hz, 1H), 3.89 (s, 3H), 1.52 (s, 9H). LCMS(m/z): 251.1 (M+1).

Step 4:(S)-tert-butyl(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)phenyl)carbamate

A mixture of methyl 3-((tert-butoxycarbonyl)amino)benzoate (500 mg, 2mmol) and

-   (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (618 mg,    3 mmol) in EtOH (1 mL) was heated at 120° C. for 3 h in a microwave    reactor under N₂. The reaction solution was concentrated and the    residue was purified by column chromatography to give the title    product (500 mg, Yield 58.8%). LCMS (m/z): 426.2 (M+1).

Step 5:(S)-3-amino-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)benzamide

To a solution of(S)-tert-butyl-(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxylpropyl)carbamoyl)phenyl)carbamate (500 mg, 1.18 mmol) in DCM (8 mL) wasadded TFA (8 mL). The mixture was stirred at 25° C. for 16 h. Thereaction solution was concentrated to give the crude title product thatwas used without further purification (400 mg). LCMS (m/z): 326.2 (M+1).

Step 6:(S)—N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-((tetrahydro-2H-pyran-4-yl)amino)benzamide

To a solution of(S)-3-amino-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxylpropyl)benzamide(400 mg, 1.23 mmol) in MeOH (8 mL) was added AcOH (0.05 mL) anddihydro-2H-pyran-4(3H)-one (123 mg, 1.23 mmol). The mixture was stirredat 25° C. for 2 h. NaBH₃CN (387 mg, 6.15 mmol) was added and theresulting mixture was stirred at 25° C. for 2 h. The reaction solutionwas concentrated, and the residue was washed with water and extractedwith EA. The organic layer was concentrated, and the residue waspurified by prep-HPLC to give the title compound (160 mg, Yield 31.8%).¹H NMR (CD₃OD, 400 MHz): δ 7.94-7.76 (m, 2H), 7.63-7.56 (m, 1H),7.56-7.49 (m, 1H), 7.32-7.24 (m, 3H), 7.21-7.15 (m, 1H), 4.71-4.55 (m,1H), 4.52-4.28 (m, 2H), 4.05-3.95 (m, 2H), 3.92-3.70 (m, 2H), 3.62-3.46(m, 3H), 3.46-3.33 (m, 4H), 3.28-3.02 (m, 2H), 1.99-1.85 (m, 2H),1.82-1.66 (m, 2H). LCMS (m/z): 410.2 (M+1).

Compound 45N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-((tetrahydrofuran-3-yl)amino)benzamide

Step 1: 2-(oxiran-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline

To a solution of 1,2,3,4-tetrahydroisoquinoline (15 g, 0.11 mol) in MeCN(100 mL) was added K₂CO₃ (30.7 g, 0.23 mol) at 0° C.2-(bromomethyl)oxirane (17 g, 0.12 mol) was added to the reaction after1 h. The solution was stirred at 22° C. for 16 h at which time thesolids were filtered and washed with MeCN. The solution was concentratedand the residue was used in the next step without further purification(17 g, Yield 78%). LCMS (m/z): 190.1 (M+1).

Step 2: 1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol

To a solution of 2-(oxiran-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline(17 g, 0.09 mol) in EtOH (300 mL) at −78° C. was slowly bubbled NH₃ (g).The reaction mixture was then sealed and heated at 80° C. for 3 h. Thereaction mixture was concentrated and the crude product was used in nextstep without further purification (18 g, Yield 96%). LCMS (m/z): 207.1(M+1).

Step 3: tert-butyl(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)phenyl)carbamate

To a solution of 3-((tert-butoxycarbonyl)amino)benzoic acid (2.5 g, 10.5mmol) in DCM (25 mL) was added EDCI (3.0 g, 15.7 mmol), HOBt (2.1 g,15.7 mmol), Et₃N (2.1 g, 21 mmol) and1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (2.2 g, 10.5mmol). The mixture was stirred at 25° C. for 16 h. The reaction solutionwas washed with water and extracted with DCM. The organic layer wasconcentrated, and the residue was purified by column chromatography togive the title product (3.2 g, 71%). LCMS (m/z): 426.3 (M+1).

Step 5:3-amino-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)benzamide

To a solution of tert-butyl(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)phenyl)carbamate(500 mg, 1.18 mmol) in DCM (5 mL) was added TFA (5 mL). The mixture wasstirred at 25° C. for 16 h. The reaction solution was concentrated, andthe residue was dissolved in water, the pH was adjusted to 7˜7.5 withsaturated aqueous of NaHCO₃ and extracted with EA. The organic layer wasconcentrated to give the title product that was used in the next stepwithout further purification (450 mg). LCMS (m/z): 326.2 (M+1).

Step 6:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-((tetrahydrofuran-3-yl)amino)benzamide

To a solution of3-amino-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)benzamide(100 mg, 0.31 mmol) in MeOH (5 mL) was added AcOH (0.05 mL) anddihydrofuran-3(2H)-one (27 mg, 0.31 mmol). The mixture was stirred at22° C. for 2 h. NaBH₃CN (98 mg, 1.55 mmol) was added and the resultingmixture was stirred at 22° C. for 2 h. The reaction solution wasconcentrated, and the residue was washed with water, extracted with EA,the organic layer was concentrated, and the residue was purified byprep-HPLC to give the title compound (22 mg, Yield 18.0%). ¹H NMR(CD₃OD, 400 MHz): δ 7.14-6.95 (m, 7H), 6.80-6.71 (m, 1H), 4.14-4.03 (m,2H), 3.99-3.89 (m, 2H), 3.87-3.78 (m, 1H), 3.75-3.69 (m, 2H), 3.67-3.61(m, 1H), 3.55-3.41 (m, 2H), 2.91-2.79 (m, 4H), 2.71-2.57 (m, 2H),2.32-2.19 (m, 1H), 1.91-1.79 (m, 1H). LCMS (m/z): 396.2 (M+1).

Compound 46N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(morpholine-4-carbonyl)benzamide

Step 1: methyl 3-(morpholine-4-carbonyl)benzoate

To a solution of 3-(methoxycarbonyl)benzoic acid (200 mg, 1.11 mmol) inDCM (10 mL) were added morpholine (200 mg, 2.30 mmol) and TEA (300 mg,2.96 mmol) and the resulting solution was stirred for 10 min at 20° C.To the mixture was added HATU (500 mg, 1.31 mmol) and the reactionmixture was stirred at 20° C. for 1 h. The mixture was concentrated andthe residue was purified via column chromatography to obtain the titleproduct (250 mg, Yield 90.5%). LCMS (m/z): 250.1 (M+1).

Step 2: 3-(morpholine-4-carbonyl)benzoic acid

To a solution of methyl methyl-3-(morpholine-4-carbonyl)benzoate (300mg, 1.11 mmol) in MeOH (2 mL) and water (2 mL) was added LiOH (100 mg,2.38 mmol) at 20° C. The mixture was heated to 60° C. for 1 h under N₂.The reaction solution was concentrated in vacuo and diluted with water.The pH was adjusted to 4 with 2N HCl and the aqueous layer was extractedwith DCM. The organic layer was concentrated to dryness and obtained thetitle product that was used in the next reaction without furtherpurification (250 mg, Yield 96%). LCMS (m/z): 236.2 (M+1).

Step 3:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(morpholine-4-carbonyl)benzamide

To a solution of 3-(morpholine-4-carbonyl)benzoic acid (300 mg crude,0.48 mmol) in MeCN (5 mL) was added1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)-propan-2-ol (100 mg, 0.49mmol), and TEA (250 mg, 2.48 mmol) and the resulting mixture was stirredat 20° C. for 10 min. BOPCl (120 mg, 0.49 mmol) was added and thereaction mixture was stirred at 20° C. for 1 h. After evaporation of thesolvent, the residue was purified by prep-HPLC to give the titlecompound (15.2 mg, Yield 7.5%). ¹H NMR (CD₃OD, 400 MHz): δ 7.85-7.94 (m,2H), 7.60 (d, J=7.8 Hz, 1H), 7.50 (t, J=7.7 Hz, 1H), 7.08-7.16 (m, 3H),7.01-7.07 (m, 1H), 4.14 (quin, J=6.0 Hz, 1H), 3.76 (s, 6H), 3.54-3.68(m, 3H), 3.47 (dd, J=6.8, 13.6 Hz, 3H), 2.80-2.98 (m, 4H), 2.63-2.74 (m,2H). LCMS (m/z): 424.2 (M+1).

Compound 49N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(methyl(tetrahydro-2H-pyran-4-yl)amino)benzamide

Step 1:tert-butyl-(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)phenyl)carbamate

To a solution of 3-((tert-butoxycarbonyl)amino)benzoic acid (2.5 g, 10.5mmol) in DCM (25 mL) was added EDCI (3.0 g, 15.7 mmol), HOBt (2.1 g,15.7 mmol), Et₃N (2.1 g, 21 mmol) and1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (2.2 g, 10.5mmol). The mixture was stirred at 25° C. for 16 h. The reaction solutionwas washed with water and extracted with DCM. The organic layer wasconcentrated, and the residue was purified by column chromatography togive the title product (3.2 g, Yield 71%). LCMS (m/z): 426.3 (M+1).

Step 3:3-amino-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)benzamide

To a solution of tert-butyl(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)phenyl)carbamate (500 mg, 1.18 mmol) in DCM (5 mL) was addedTFA (5 mL). The mixture was stirred at 25° C. for 16 h. The reactionsolution was concentrated, and the residue was dissolved in water, thepH was adjusted to 7˜7.5 with saturated aqueous of NaHCO₃ and extractedwith EA. The organic layer was concentrated to give the title productthat was used in the next step without further purification (450 mg,crude). LCMS (m/z): 326.2 (M+1).

Step 4.N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(methyl(tetrahydro-2H-pyran-4-yl)amino)benzamide

To a solution ofN-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-((tetrahydro-2H-pyran-4-yl)amino)benzamide(300 mg, 0.73 mmol) in MeOH (6 mL) was added AcOH (0.05 mL) and HCHO(548 mg, 7.3 mmol, 40% w/w). The mixture was stirred at 20° C. for 2 h.NaBH₃CN (276 mg, 4.38 mmol) was added and the resulting mixture wasstirred at 20° C. for 16 h. The reaction solution was concentrated, theresidue was washed with water and extracted with EA. The organic layerwas concentrated, and the residue was purified by prep-HPLC to give thetitle compound (105 mg, Yield 33.9%). ¹H NMR (CD₃OD, 400 MHz): δ7.37-7.29 (m, 1H), 7.23-7.17 (m, 1H), 7.17-6.97 (m, 6H), 4.18-4.08 (m,1H), 4.07-3.90 (m, 3H), 3.80-3.68 (m, 2H), 3.62-3.51 (m, 3H), 3.51-3.43(m, 1H), 2.99-2.79 (m, 7H), 2.75-2.58 (m, 2H), 1.94-1.79 (m, 2H),1.72-1.59 (m, 2H). LCMS (m/z): 424.1 (M+1).

Compound 50N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(oxetan-3-ylamino)benzamide

Step 1:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(oxetan-3-ylamino)benzamide

To a solution of3-amino-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)benzamide(100 mg, 0.31 mmol) in MeOH (5 mL) was added AcOH (0.05 mL) andoxetan-3-one (22 mg, 0.31 mmol). The mixture was stirred at 22° C. for 2h. NaBH₃CN (98 mg, 1.55 mmol) was added, and the resulting mixture wasstirred at 22° C. for 2 h. The reaction solution was concentrated, theresidue was washed with water, extracted with EA, the organic layer wasconcentrated, and the residue was purified by prep-HPLC to give thetitle compound (17 mg, Yield 14.4%). ¹H NMR (CD₃OD, 400 MHz): δ7.17-6.97 (m, 6H), 6.96-6.88 (m, 1H), 6.72-6.62 (m, 1H), 5.03-4.95 (m,2H), 4.67-4.59 (m, 1H), 4.59-4.49 (m, 2H), 4.15-4.04 (m, 1H), 3.80-3.69(m, 2H), 3.56-3.40 (m, 2H), 2.96-2.79 (m, 4H), 2.73-2.58 (m, 2H). LCMS(m/z): 382.2 (M+1).

Compound 51N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(morpholinomethyl)benzamide

Step 1: methyl 3-(morpholinomethyl)benzoate

To a solution of methyl 3-formylbenzoate (100 mg, 0.61 mmol) in MeOH (5mL) was added morpholine (100 mg, 1.15 mmol) and the resulting mixturewas stirred for 10 min at 20° C. To the mixture was added NaBH₃CN (100mg, 1.59 mmol) and the reaction mixture was stirred for 30 min at 20° C.The solution was concentrated and the residue was purified by prep-TLCto afford the title compound (130 g, Yield 90.9%). ¹H NMR (CD₃OD, 400MHz): δ 7.98-8.05 (m, 1H), 7.92 (td, J=1.4, 7.7 Hz, 1H), 7.57-7.62 (m,1H), 7.37-7.53 (m, 1H), 3.90 (s, 3H), 3.66-3.71 (m, 4H), 3.57 (s, 2H),2.41-2.49 (m, 4H).

Step 2: 3-(morpholinomethyl)benzoic acid

To a solution of methyl 3-(morpholinomethyl)benzoate (150 mg, 0.64 mmol)in MeOH (2 mL) and water (2 mL) was added LiOH (55 mg, 1.31 mmol) at 20°C. The mixture was heated to 60° C. for 1 h. The reaction solution wasconcentrated and purified via prep-HPLC to give the title compound (60mg, Yield 42.5%). LCMS (m/z): 222 (M+1).

Step 3:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-(morpholinomethyl)benzamide

To a solution of 3-(morpholinomethyl)benzoic acid (60 mg, 0.27 mmol) inMeCN (3 mL) were added1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (60 mg, 0.29mmol), TEA (70 mg, 0.69 mmol) and the resulting mixture was stirred at20° C. for 10 min. BOPCl (70 mg, 0.28 mmol) was added and the reactionmixture was stirred at 20° C. for 16 h. The reaction solution wasconcentrated and the residue was purified by prep-HPLC to give the titlecompound (4 mg, Yield 3.6%). ¹H NMR (CD₃OD, 400 MHz): δ 7.77 (s, 1H),7.68 (d, J=7.8 Hz, 1H), 7.50 (d, J=7.7 Hz, 1H), 7.35 (t, J=7.7 Hz, 1H),7.07-7.14 (m, 3H), 7.00-7.06 (m, 1H), 4.11 (quin, J=6.0 Hz, 1H), 3.75(s, 2H), 3.64-3.72 (m, 4H), 3.43-3.59 (m, 4H), 2.83-2.93 (m, 4H),2.61-2.74 (m, 2H), 2.44 (brs, 4H). LCMS (m/z): 410.1 (M+1).

Compound 52N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-((1-(tetrahydro-2H-pyran-4-yl)ethyl)amino)benzamide

Step 1:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-((1-(tetrahydro-2H-pyran-4-yl)ethyl)amino)benzamide

A solution of3-amino-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)benzamide(130 mg, 0.4 mmol), 1-(tetrahydro-2H-pyran-4-yl)ethanone (52 mg, 0.4mmol) and AcOH (0.1 mL) in MeOH (10 mL). The mixture was stirred at 22°C. for 1 h, then NaBH₃CN (76 mg, 1.2 mmol) was added. The mixture wasstirred at 22° C. for 4 h. The reaction mixture was concentrated andquenched with water. The mixture solution was extracted with DCM, thecombined organic layers were concentrated and the residue was purifiedby prep-TLC to give the desired compound (14.0 mg, Yield 8%). ¹H NMR(CD₃OD, 400 MHz): δ 6.90-7.07 (m, 6H), 6.81 (d, J=7.5 Hz, 1H), 6.64 (d,J=8.03 Hz, 1H), 3.98-4.04 (m, 1H), 3.87 (d, J=11.3 Hz, 2H), 3.70 (s,2H), 3.28-3.45 (m, 5H), 2.82 (brs, 4H), 2.56-2.65 (m, 2H), 1.70 (d,J=13.8 Hz, 1H), 1.57 (brs, 1H), 1.17-1.37 (m, 3H), 1.04 (d, J=6.3 Hz,3H). LCMS (m/z): 438.3 (M+1).

Compound 53N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-((2,2-dimethyltetrahydro-2H-pyran-4-yl)amino)benzamide

Step 1:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-((2,2-dimethyltetrahydro-2H-pyran-4-yl)amino)benzamide

A solution of3-amino-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)benzamide(130 mg, 0.4 mmol), 2,2-dimethyldihydro-2H-pyran-4(3H)-one (52 mg, 0.4mmol) and AcOH (0.1 mL) in MeOH (10 mL). The mixture was stirred at 22°C. for 12 h, then NaBH₃CN (76 mg, 1.2 mmol) was added and the resultingmixture was stirred at 22° C. for 2 h. The reaction mixture wasconcentrated and quenched with water. The aqueous mixture was extractedwith DCM, the combined organic layers were concentrated and the residuewas purified by prep-HPLC to give the title compound (5.5 mg, Yield3.1%). ¹H NMR (CD₃OD, 400 MHz): δ 8.42 (brs, 1H) 7.00-7.33 (m, 7H) 6.84(d, J=7.8 Hz, 1H) 4.21-4.41 (m, 3H) 3.40-3.92 (m, 8H) 3.11-3.20 (m, 3H)1.91-2.07 (m, 2H) 1.18-1.44 (m, 8H). LCMS (m/z): 438.3 (M+1).

Compound 54N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-((tetrahydro-2H-pyran-4-yl)amino)nicotinamide

Step 1.5-bromo-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)nicotinamide

A solution of 1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol(326 mg, 1.58 mmol), 5-bromonicotinic acid (300 mg, 1.5 mmol), HATU (627mg, 1.65 mmol) and TEA (181.8 mg, 1.8 mmol) in DCM (15 mL) was stirredat 22° C. for 2 h, at which time the reaction mixture was diluted withwater and extracted with DCM. The combined organic layers were dried andconcentrated and the residue was purified by column chromatography togive title compound that was used in the next step without furtherpurification (200 mg, Yield 34%). LCMS (m/z): 390/392 (M+1/M+2).

Step 2:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-((tetrahydro-2H-pyran-4-yl)amino)nicotinamide

To a solution of5-bromo-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)nicotinamide(100 mg, 0.26 mmol) in dioxane (10 mL) were addedtetrahydro-2H-pyran-4-amine (39.4 mg, 0.29 mmol), Pd₂(dba)₃ (20 mg, 0.02mmol), NaOtBu (24 mg, 0.52 mmol) and BINAP (26 mg, 0.04 mmol). Thereaction mixture was heated at 110° C. for 6 h under N₂. The mixture wasconcentrated and the residue was dissolved in EA, washed with water, theorganic layer was collected, dried, and the residue purified byprep-HPLC to give the title compound (25.9 mg, Yield 24%). ¹H NMR(CD₃OD, 400 MHz): δ 8.17 (d, J=1.5 Hz, 1H), 8.05 (d, J=2.5 Hz, 1H),7.43-7.35 (m, 1H), 7.13-7.06 (m, 3H), 7.04-6.99 (m, 1H), 4.17-4.07 (m,1H), 4.02-3.93 (m, 2H), 3.73 (s, 2H), 3.63-3.50 (m, 4H), 3.41 (dd,J=6.8, 13.6 Hz, 1H), 2.94-2.82 (m, 4H), 2.70-2.57 (m, 2H), 2.03-1.93 (m,2H), 1.57-1.45 (m, 2H). LCMS (m/z): 411.1 (M+1).

Compound 55N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4-((tetrahydro-2H-pyran-4-yl)amino)picolinamide

Step 1:methyl 4-((tetrahydro-2H-pyran-4-yl)amino)picolinate

To a solution of compound methyl 4-chloropicolinate (100 mg, 0.59 mmol),tetrahydro-2H-pyran-4-amine hydrochloride (121 mg, 0.88 mmol), Cs₂CO₃(762 mg, 2.34 mmol), Pd₂(dba)₃ (54 mg, 0.059 mmol) and XPhos (28 mg,0.06 mmol) in toluene (10 mL) was stirred and heated at 110° C. under N₂for 16 h. The catalyst was filtered and the filtrate was washed with EA,concentrated in vacuo and the residue was purified by prep-TLC to givethe title product (50 mg, Yield 36.2%). LCMS (m/z): 237.2 (M+1).

Step 2:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4-((tetrahydro-2H-pyran-4-yl)amino)picolinamide

To a solution of compound methyl4-((tetrahydro-2H-pyran-4-yl)amino)picolinate (50 mg, 0.21 mmol) and1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (44 mg, 0.21mmol) in MeOH (2 mL) was stirred at 100° C. under microwave heating for3 h. The reaction mixture was purified by prep-HPLC to give the titlecompound (29.9 mg, Yield 34.4%). ¹H NMR (400 MHz, CD₃OD): δ 7.95 (d,J=5.6 Hz, 1H), 7.26 (d, J=2.4 Hz, 1H), 7.13-7.05 (m, 3H), 7.03-6.98 (m,1H), 6.62 (dd, J=2.4, 5.6 Hz, 1H), 4.07 (quin, J=6.0 Hz, 1H), 4.01-3.93(m, 2H), 3.72 (s, 2H), 3.66-3.60 (m, 1H), 3.60-3.57 (m, 1H), 3.57-3.52(m, 2H), 3.49-3.42 (m, 1H), 2.95-2.89 (m, 2H), 2.87-2.81 (m, 2H), 2.65(d, J=6.0 Hz, 2H), 1.97 (d, J=12.8 Hz, 2H), 1.59-1.47 (m, 2H). LCMS(m/z): 411.1 (M+1).

Compound 57N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-fluoro-5-((tetrahydro-2H-pyran-4-yl)amino)benzamide

Step 1:5-amino-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-fluorobenzamide

The solution of 5-amino-2-fluorobenzoic acid (200 mg, 1.29 mmol) andHATU (490 mg, 1.29 mmol) in DCM (15 mL) was stirred at 17° C. for 30min. Then 1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (265mg, 1.29 mmol) and DIPEA (333 mg, 2.58 mmol) was added and the resultingsolution was stirred at 17° C. for 16 h. The solution was concentratedand the residue was purified by column chromatography to give desiredproduct (372 mg, Yield 84%).

Step 2:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-fluoro-5-((tetrahydro-2H-pyran-4-yl)amino)benzamide

A solution of5-amino-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-fluorobenzamide(372 mg, 1.08 mmol), dihydro-2H-pyran-4(3H)-one (108 mg, 1.08 mmol) andAcOH (0.05 mL) in MeOH (20 mL) was stirred at 17° C. for 2 h. ThenNaBH₃CN (109 mg, 1.63 mmol) was added and the resulting solution wasstirred at 17° C. for 4 h. The solution was concentrated and the residuewas purified by column chromatography to afford the title product (121.5mg, Yield 17.5%). ¹H NMR (CD₃OD, 400 MHz): δ 7.16-7.07 (m, 3H), 7.03(dd, J=2.9, 5.9 Hz, 2H), 6.95 (dd, J=8.9, 10.7 Hz, 1H), 6.79 (td, J=3.6,8.8 Hz, 1H), 4.11 (quin, J=6.0 Hz, 1H), 3.98 (d, J=11.5 Hz, 2H),3.80-3.69 (m, 2H), 3.64-3.40 (m, 5H), 2.98-2.81 (m, 4H), 2.72-2.60 (m,2H), 1.99 (d, J=12.8 Hz, 2H), 1.56-1.39 (m, 2H). LCMS (m/z): 428.2(M+1).

Compound 58N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4-((tetrahydro-2H-pyran-4-yl)oxy)benzamide

Step 1: methyl 4-((tetrahydro-2H-pyran-4-yl)oxy)benzoate

To a solution of ethyl 4-hydroxybenzoate (500 mg, 3.0 mmol),tetrahydro-2H-pyran-4-ol (307.3 mg, 3.0 mmol) and PPh₃ (944 mg, 3.6mmol) in THF (15 mL) was added DEAD (627 mg, 3.6 mmol) at 0° C. Themixture was the warmed to 21° C. and stirred for 16 h. The mixture wastreated with water and the organic layer was washed with brine, driedover Na₂SO₄, concentrated and the residue was purified by columnchromatography to give the title compound (320 mg, Yield 45%). ¹H NMR(CDCl₃, 400 MHz): δ 7.92 (d, J=8.9 Hz, 1H), 6.75-6.94 (m, 1H), 4.44-4.59(m, 1H), 4.28 (d, J=7.2 Hz, 2H), 3.83-4.00 (m, 2H), 3.46-3.60 (m, 2H),1.88-2.05 (m, 2H), 1.67-1.83 (m, 2H), 1.31 (t, J=7.2 Hz, 3H).

Step 2: 4-((tetrahydro-2H-pyran-4-yl)oxy)benzoic acid

To a solution of methyl 4-((tetrahydro-2H-pyran-4-yl)oxy)benzoate (400mg, 1.6 mmol) in MeOH (10 ml) was added a solution of NaOH (128 mg, 3.2mmol) in H₂O (4 mL) at 22° C. The mixture was stirred at 50° C. for 4 h.The mixture was concentrated and the residue was treated with water andextracted with EA. The water layer was treated with 2N HCl to pH=3. Thewater layer was then extracted with EA. The organic layer was washedwith brine, dried over Na₂SO₄ and concentrated to give the title productwhich was used in next step without further purification (350 mg, Yield98.6%). ¹H NMR (CDCl₃, 400 MHz): δ 8.08 (d, J=8.9 Hz, 2H), 6.98 (d,J=8.9 Hz, 2H), 4.64 (tt, J=7.7, 3.8 Hz, 1H), 3.95-4.09 (m, 2H), 3.64(ddd, J=11.6, 8.2, 3.3 Hz, 2H), 2.01-2.13 (m, 2H), 1.78-1.93 (m, 2H).

Step 3:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4-((tetrahydro-2H-pyran-4-yl)oxy)benzamide

To a solution of 4-((tetrahydro-2H-pyran-4-yl)oxy)benzoic acid (150 mg,0.67 mmol) in DMF (4 mL) was added DIEA (260 mg, 2.01 mmol), HATU (384mg, 1.01 mmol) and1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (170 mg, 0.81mmol). The reaction mixture was stirred at 22° C. for 16 h. The mixturewas treated with water and extracted with EA. The organic layer waswashed with brine, dried over Na₂SO₄, concentrated and the residue waswas purified by prep-HPLC to give the title compound (206.1 mg, Yield74.9%). ¹H NMR (CD₃OD, 400 MHz): δ 7.84 (d, J=8.8 Hz, 2H), 7.26-7.37 (m,3H), 7.19-7.25 (m, 1H), 7.05 (d, J=8.8 Hz, 2H), 4.59-4.74 (m, 2H),4.31-4.49 (m, 2H), 3.93-4.01 (m, 2H), 3.86 (brs, 1H), 3.63 (ddd, J=11.7,8.8, 3.0 Hz, 2H), 3.53 (qd, J=14.0, 5.7 Hz, 3H), 3.37-3.44 (m, 1H),3.11-3.32 (m, 3H), 2.02-2.12 (m, 2H), 1.69-1.81 (m, 2H). LCMS (m/z):411.2 (M+1).

Compound 166(S)—N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-6-(oxetan-3-ylamino)pyrimidine-4-carboxamide

Step 1: 6-Hydroxypyrimidine-4-carboxylic acid

To a solution of sodium (Z)-1,4-diethoxy-1,4-dioxobut-2-en-2-olate (55.0g, 262 mmol) in H₂O (500 mL) was added formimidamide acetate (27.3 g,262 mmol) and NaOH (10.5 g). After addition, the resulting mixture wasstirred at 25° C. for 16 h then concentrated and then acidified by addedaqueous HCl (1N) until pH=1. The resulting solid was collected byfiltration, washed with H₂O and ether to give6-hydroxypyrimidine-4-carboxylic acid (6.0 g, yield: 16.3%). ¹H NMR (400MHz, DMSO-d₆) δ 12.89 (s, 1H), 8.24 (s, 1H), 6.83 (s, 1H).

Step 2: 6-chloropyrimidine-4-carboxylic acid

To a solution of 6-hydroxypyrimidine-4-carboxylic acid (6.0 g, 42.8mmol) in EtOAc (90 mL) was added (COCl)₂ (12 mL) dropwise, followed by afew drops of DMF. The mixture was stirred at 75° C. for 3 h, and then at25° C. for 16 h. The solvent was evaporated to give the crude6-chloropyrimidine-4-carboxylic acid (6.3 g, yield: 92.9%). ¹H NMR (400MHz, DMSO-d₆) δ 8.31 (s, 1H), 6.88 (s, 1H).

Step 3: 6-chloropyrimidine-4-carbonyl chloride

A drop of DMF was added to a stirred solution of6-chloropyrimidine-4-carboxylic acid (5.5 g, 34.7 mmol) and (COCl)₂ (12mL) in DCM (100 mL). The mixture was stirred at 25° C. for 2 h. Thesolvent was evaporated under reduced pressure to give crude6-chloropyrimidine-4-carbonyl chloride (6.0 g, yield: 97.7%). ¹H NMR(400 MHz, DMSO-d₆) δ 9.20 (s, 1H), 8.10 (s, 1H).

Step 4:(S)-6-chloro-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide

To a stirred and cooled (0° C.) solution of(S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (7.15 g, 34.7mmol) and Et₃N (14.0 g, 138.8 mmol) in DCM (100 mL) was added6-chloropyrimidine-4-carbonyl chloride (5.5 g, 34.7 mmol). Afteraddition, the resulting mixture was stirred at 25° C. for 16 h, at whichtime LCMS showed the completion of the reaction. The solvent wasevaporated and the residue purified by flash chromatography to give the(S)-6-chloro-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide(7.2 g, yield: 60%). LCMS (m/z): 347.0 [M+H]⁺

Step 5:(S)—N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-6-(oxetan-3-ylamino)pyrimidine-4-carboxamide

To a solution of(S)-6-chloro-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide (347 mg, 1 mmol) in i-PrOH (5 mL) wasadded oxetan-3-amine (73.1 mg, 1 mmol) and DIPEA (129 mg, 1 mmol). Theresulting mixture was stirred at 110° C. for 16 hours, at which timeLCMS showed the completion of the reaction. After evaporation of thesolvent, the residue was purified by preparative HPLC to give the targetcompound(S)—N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-6-(oxetan-3-ylamino)pyrimidine-4-carboxamide(62.5 mg, yield: 16.3%). ¹H NMR (400 MHz, MeOD-d₄) δ 8.24 (s, 1H),7.15-7.05 (m, 4H), 7.02-6.98 (m, 1H), 5.09 (s, 1H), 4.95 (t, J=6.8 Hz,2H), 4.59 (t, J=6.3 Hz, 2H), 4.10-4.03 (m, 1H), 3.72 (s, 2H), 3.56-3.46(m, 2H), 2.96-2.91 (m, 2H), 2.87-2.80 (m, 2H), 2.65 (d, J=6.3 Hz, 2H).LCMS (m/z): 384.1 [M+H]⁺.

Compound 84N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-methylquinoline-6-carboxamide

Step 1: 2-(oxiran-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline

To a stirring solution of 1,2,3,4-tetrahydroisoquinoline (15 g, 0.11mol) in MeCN (100 mL) at 0° C. was added K₂CO₃ (30.7 g, 0.23 mol), then2-(bromomethyl)oxirane (17 g, 0.12 mol) added slowly over a period of 1h. After the addition the solution was stirred at 21° C. for 12 h. Theresulting solid was then removed by filtration and washed with MeCN andthe combined organic filtrate was concentrated under reduced pressure togive the crude product. This residue was used into next step withoutfurther purification (17 g, Yield: 78%). LCMS (m/z): 190.1 (M+1).

Step 2: 1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol

NH₃ was bubbled into a stirred and cooled (−78° C.) solution of2-(oxiran-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline (17 g, 0.09 mol) inEtOH (300 mL). After saturation, the reaction mixture was then sealedand heated at 80° C. for 3 h. After LCMS indicated the reaction to becomplete, the reaction mixture was concentrated and the crude productused in the next step without further purification (18 g, Yield 96%).LCMS (m/z): 207.1 (M+1).

Step 3:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-methylquinoline-6-carboxamide

To a solution of 2-methylquinoline-6-carboxylic acid (100 mg, 0.535mmol) in DCM (20 mL) was added HATU (244 mg, 0.642 mmol) and TEA (162mg, 1.604 mmol). The mixture was stirred at 15° C. for 30 minutes before1-amino-3-(3,4-dihydro isoquinolin-2(1H)-yl)propan-2-ol (110 mg, 0.535mmol) was added. The resulting mixture was stirred for another 16 h at15° C., at which point LCMS showed the completion of the reaction. Themixture was concentrated and the residue was purified by PreparationHPLC to give the desired title compound (106.2 mg, 53%). ¹H NMR (400MHz, METHANOL-d₄) δ=8.32 (d, J=1.9 Hz, 1H), 8.14 (d, J=8.4 Hz, 1H), 8.09(dd, J=2.1, 8.8 Hz, 1H), 7.93 (d, J=8.8 Hz, 1H), 7.49 (d, J=8.4 Hz, 1H),7.16-7.08 (m, 3H), 7.08-7.03 (m, 1H), 4.18 (quin, J=6.1 Hz, 1H), 3.79(s, 2H), 3.59 (d, J=5.8 Hz, 2H), 2.94-2.88 (m, 4H), 2.79-2.68 (m, 5H).LCMS (m/z): 376.0 (M+1).

Compound 219(R)—N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-methylquinoline-6-carboxamide

To a solution of 2-methylquinoline-6-carboxylic acid (200 mg, 1.070mmol) in DCM (30 mL), was added HATU (489 mg, 1.283 mmol) and TEA (324mg, 3.208 mmol). The solution was stirred at 15° C. for 30 minutesbefore (R)-1-amino-3-(3,4-dihydro isoquinolin-2(1H)-yl)propan-2-ol (264mg, 1.283 mmol) was added. The resulting solution was stirred foranother 16 h at 15° C., until the reaction was complete by LCMSanalysis. The mixture was then concentrated under vacuum to give thecrude material which was purified by Preparative HPLC to give the titlecompound (118 mg, 29%). ¹H NMR (400 MHz, METHANOL-d₄) δ 8.33 (d, J=1.9Hz, 1H), 8.15 (d, J=8.5 Hz, 1H), 8.09 (dd, J=2.1, 8.8 Hz, 1H), 7.93 (d,J=8.8 Hz, 1H), 7.49 (d, J=8.4 Hz, 1H), 7.15-7.05 (m, 4H), 4.18 (quin,J=6.1 Hz, 1H), 3.79 (s, 2H), 3.63-3.55 (m, 2H), 2.95-2.90 (m, 4H), 2.76(s, 3H), 2.76-2.68 (m, 2H). LCMS (m/z): 376.1 [M+H]⁺

Compound 221(S)—N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-methylquinoline-6-carboxamide

To a solution of 2-methylquinoline-6-carboxylic acid (1 g, 5.35 mmol) inDCM (100 mL), was added HATU (2.44 g, 6.42 mmol) and TEA (1620 mg,16.043 mmol). The solution was stirred at 15° C. for 30 minutes before(S)-1-amino-3-(3,4-dihydro isoquinolin-2(1H)-yl)propan-2-ol (1.76 g,8.55 mmol) was added. The resulting solution was stirred for 16 h at 15°C. until LCMS analysis showed the reaction to be complete. The mixturewas then concentrated under vacuum and the residue purified byPreparative HPLC to give the desired title compound (502.1 mg, 25%). ¹HNMR (400 MHz, METHANOL-d₄) δ 8.31 (br. s., 1H), 8.08 (d, J=8.8 Hz, 1H),8.12 (d, J=8.4 Hz, 1H), 7.91 (d, J=8.8 Hz, 1H), 7.47 (d, J=8.3 Hz, 1H),7.17-7.03 (m, 4H), 4.23-4.11 (m, 1H), 3.78 (br. s., 2H), 3.59 (d, J=5.5Hz, 2H), 2.91 (br. s., 4H), 2.78-2.69 (m, 5H). LCMS (m/z): 376.1 [M+H]⁺.

Compound 208(S)-6-((1-acetylpiperidin-4-yl)amino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide

Step 1: tert-butyl (1-acetylpiperidin-4-yl)carbamate

To a solution of tert-butyl piperidin-4-ylcarbamate (200 g, 1 mol) andEt₃N (150 g, 1.5 mol) in DCM (3000 mL) was added Ac₂O (102 g, 1 mol)dropwise over 1 h, while maintained the temperature at 0° C. Afteraddition, the mixture was stirred 0° C. for another 2 h, at which timeTLC showed the reaction was completed. The solution was quenched byaddition of water (1 L). The organic phase was collected and washed withsaturated aqueous NaHCO₃ (1 L), dried (Na₂SO₄) and concentrated to givecrude product. Four batches were run in parallel and produced a combinedcrude product weight of 670 g. This crude was used directly in the step.LCMS (m/z): 243.1 (M+1).

Step 2: 1-(4-aminopiperidin-1-yl)ethanone hydrochloride

To a solution of tert-butyl (1-acetylpiperidin-4-yl)carbamate (330 g,1.36 mol) in MeOH (1000 mL) was added HCl/MeOH (4M, 300 mL) over 30 minto maintain the temperature at 0° C. After addition, the mixture wasstirred at 0° C. for another 2 h and then concentrated to give the crudeproduct. Two batches were run in parallel and produced a combined crudeproduct weight of 310 g. This crude was used in next step withoutfurther purification. ¹H NMR (400 MHz, D₂O) δ 4.35 (dd, J=2.0, 12.0 Hz,1H), 3.98-3.85 (m, 1H), 3.44-3.30 (m, 1H), 3.18-3.05 (m, 1H), 2.75-2.58(m, 1H), 2.06-1.92 (m, 5H), 1.61-1.31 (m, 2H); LCMS (m/z): 143.1 (M+1).

Step 3: 6-chloropyrimidine-4-carbonyl chloride

To a stirred mixture of 6-hydroxypyrimidine-4-carboxylic acid (300 g,2.14 mol) in EA (3000 mL), oxalyl dichloride (1356 g, 10.68 mol) wasdropped slowly to maintain a reaction temperature below 30° C. Afteraddition, the mixture was stirred at 20° C. for 30 min and then 2 mL ofDMF was added to the mixture. The mixture was then stirred at 80° C. for16 hours and concentrated to give the crude product as black solid.Three batches were run in parallel and produced a combined crude productweight of 787 g. This crude was used directly in the next step.

Step 4:(S)-6-chloro-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide

To a stirred mixture of(S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (247 g, 1.20mol), and TEA (250 g, 2.5 mol) in DCM (3500 mL) was added6-chloropyrimidine-4-carbonyl chloride (190 g in 100 mL of DCM) slowlyat −60° C. over 1 h. After addition, the mixture was then allowed towarm to 10° C. Stirring was continued for 1 h, at which time TLC showedthe reaction was completed. The reaction was quenched by addition ofwater (1.5L). The organic phase was collected, dried (Na₂SO₄) andevaporated. The residue was purified by flash chromatography(EtOAc˜DCM:MeOH=10:1) to give the desired product as a pale yellowsolid. Four batches were run in parallel and produced a combined crudeproduct weight of 800 g, 49% yield. ¹H NMR (400 MHz, MeOD-d4) δ 8.73 (d,J=1.0 Hz, 1H), 8.07 (d, J=1.0 Hz, 1H), 7.17-7.06 (m, 3H), 7.00 (d, J=7.0Hz, 1H), 4.12 (q, J=6.0 Hz, 1H), 3.74 (s, 2H), 3.64-3.53 (m, 2H), 2.94(q, J=5.5 Hz, 2H), 2.92-2.81 (m, 2H), 2.78-2.64 (m, 2H); LCMS (m/z):347.2 [M+H]⁺

Step 5:(S)-6-((1-acetylpiperidin-4-yl)amino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide

The solution of(S)-6-chloro-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide(190 g, 0.55 mmol) and 1-(4-aminopiperidin-1-yl)ethanone (78 g), Et₃N(100 g, 1 mol) in i-PrOH (2000 mL) was stirred at 60° C. for 16 h, atwhich time LCMS showed completed conversion. The mixture wasconcentrated and the residue was purified by flash chromatography togive the crude product. Four batches were run in parallel and produced acombined crude product weight of 482 g. This crude was further purifiedon preparative HPLC to give the title compound (325 g, >98% purity, freebase form). ¹H NMR (400 MHz, MeOD-d4) 8.26 (s, 1H), 7.15-7.02 (m, 5H),4.46 (m, 1H), 4.15-4.07 (m, 2H), 3.88 (m, 1H), 3.74 (s, 2H), 3.53 (m,2H), 3.33 (m, 1H), 2.95-2.86 (m, 5H), 2.68 (m, 2H), 2.14-2.01 (m, 5H),1.48-1.42 (m, 2H); LCMS (m/z): 453.3 [M+H]⁺

Step 6:(S)-6-((1-acetylpiperidin-4-yl)amino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamidehydrochloride

The free base was dissolved in DCM (100 mL) and added dropwise to astirred and cooled solution of HCl (6N in EtOAc, 1 L) at −30° C.Stirring at −30° C. was continued for another 1 h and the resultingprecipitate was collected by filtration. The solid was washed with DCMand EtOAc, dried to give the HCl salt of the target compound (301.4 g,yield: 30.2%) as a white solid. ¹H NMR (400 MHz, D₂O) δ 8.59 (s, 1H),7.30-7.17 (m, 3H), 7.17-7.07 (m, 2H), 4.55 (dd, J=6.4, 15.4 Hz, 1H),4.43-4.19 (m, 4H), 3.88 (d, J=13.8 Hz, 1H), 3.82-3.72 (m, 1H), 3.52-3.33(m, 4H), 3.31-3.08 (m, 4H), 2.86 (t, J=11.6 Hz, 1H), 2.11-1.94 (m, 5H),1.67-1.40 (m, 2H); LCMS (m/z): 453.2 [M+H]⁺.

Compound 254

(S)-2-((1-acetylpiperidin-4-yl)amino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)isonicotinamideStep 1: methyl2-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)isonicotinate

A mixture of methyl 2-bromoisonicotinate (160 g, 0.69 mol) andtert-butyl 4-amino piperidine-1-carboxylate (200 g, 1.0 mol), Pd₂(dba)₃(8 g, 5% w), xantphos (8 g, 5% w), Cs₂CO₃ (326 g, 1.0 mol) in dioxane(2500 mL) was stirred at 80° C. under N₂ for 16 h. After completion ofthe reaction, the mixture was concentrated and the residue dissolved inwater (800 mL) and extracted with DCM (1000 mL×3). The combined organiclayers were dried and concentrated. The residue was purified by flashchromatography to give the product. Nine batches were run in paralleland produced a combined product weight of 700 g, Yield: 33.4%. ¹H NMR(400 MHz, CDCl₃) δ 8.19 (d, J=5.2 Hz, 1H), 7.08 (d, J=5.2 Hz, 1H), 6.96(s, 1H), 4.62 (d, J=8.0 Hz, 1H), 4.05 (br. s., 2H), 3.92 (s, 3H), 2.97(t, J=12.0 Hz, 2H), 2.11-1.97 (m, 2H), 1.48 (s, 9H), 1.42-1.35 (m, 2H).LCMS (m/z): 336.1 (M+1).

Step 2: 2-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)isonicotinicacid

To a solution of methyl2-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)isonicotinate (230 g,0.69 mol) in MeOH (1500 mL) was added aq.NaOH (56 g, in 200 mL of water)over 20 min at 0° C. After addition, the mixture was stirred at roomtemperature for 2 h. MeOH was then removed under reduced pressure andthe aqueous solution then pH adjusted to pH=6 by acidifying with theaddition of 4N HCl. The resulting precipitate was collected byfiltration, washed with water and dried to give the crude product. Threebatches were run in parallel and produced a combined crude productweight of 590 g, yield: 89.4%. This crude was used in next step withoutfurther purification. LCMS (m/z): 322.2 (M+1).

Step 3: (S)-tert-butyl4-((4-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)pyridin-2-yl)amino)piperidine-1-carboxylate

To a solution of2-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)isonicotinic acid (150g, 0.47 mol) in DCM (1500 mL) was added HATU (178 g, 0.47 mol) and TEA(47 g, 0.47 mol) at 20° C., then the mixture was stirred at thetemperature for 2 h.(S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (113 g, 0.55mol) was added to the solution, and the mixture was stirred at 20° C.for another 16 h, at which time TLC showed the completion of thereaction. The mixture washed with water (200 mL) and the combinedorganic phases were dried and concentrated. The residue was purified byflash chromatography (EtOAc˜DCM:MeOH=10:1) to give the title compound asyellowish oil. Four batches were run in parallel and produced a combinedproduct weight of 510 g, yield: 53.2%. LCMS (m/z): 510.2 [M+H]⁺.

Step 4:(S)—N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-(piperidin-4-ylamino)isonicotinamidehydrochloride

The mixture of (S)-tert-butyl4-((4-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)pyridin-2-yl)amino)piperidine-1-carboxylate(510 g, 1.0 mol) in DCM (1000 mL) was dropped slowly into a stirred andcooled (−30° C.) solution of HCl (4M in EtOAc, 2000 mL). After addition,the mixture was stirred at −30° C. for 30 min. The resulting solid wasthen collected by filtration, washed with DCM and dried under reducedpressure to give the title compound (350 g yield: 85.4%, HCl salt) as awhite solid. LCMS (m/z): 410.2 [M+H]⁺

Step 5:(S)-2-((1-acetylpiperidin-4-yl)amino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)isonicotinamide

To a stirred mixture of(S)—N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-(piperidin-4-ylamino)isonicotinamide(70 g), and Et₃N (40 g) in DCM (2000 mL) was added Ac₂O (17 g) dropwiseover 1 h at 0° C. After addition, the mixture was warmed to 20° C. andstirring was continued for another 1 h, at which time TLC showed thereaction was completed. The reaction mixture was washed with water (500mL), and the organic phase dried and concentrated. The residue was thenpurified by flash chromatography (EtOAc˜DCM:MeOH=10:1) to give crudeproduct. Five batches were run in parallel and produced a combined crudeproduct weight of 400 g. This crude was further purified by preparativeHPLC to give the pure product (310 g, >98% purity, free base form). ¹HNMR (400 MHz, MeOD-d4) 7.94-7.92 (d, 7.0 Hz, 1H), 7.14-7.05 (m, 4H),6.87 (s, 1H), 6.76-6.74 (m, 1H), 4.44 (m, 1H), 4.10 (m, 1H), 3.96-3.94(m, 2H), 3.75 (s, 2H), 3.52 (m, 2H), 3.33-3.32 (m, 1H), 2.92-2.86 (m,5H), 2.67 (m, 2H), 2.13-2.00 (m, 5H), 1.44-1.37 (m, 2H); LCMS (m/z):452.3 [M+H]⁺

Step 6:(S)-2-((1-acetylpiperidin-4-yl)amino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)isonicotinamidehydrochloride

The free base was dissolved in DCM (100 mL) and added dropwise to astirred and cooled solution of HCl (6N in EtOAc, 1 L) at −30° C.Stirring at −30° C. was continued for another 1 h and the resultingprecipitate was collected by filtration. The solid was washed with DCMand EtOAc, dried to give the HCl salt of the product (302.2 g, yield:78.0%) as a white solid. ¹H NMR (400 MHz, MeOD-d4) δ 8.00 (d, J=6.8 Hz,1H), 7.64 (br. s., 1H), 7.36-7.18 (m, 5H), 4.70 (d, J=15.4 Hz, 1H),4.60-4.39 (m, 3H), 4.19 (br. s., 1H), 4.11 (d, J=13.2 Hz, 1H), 3.98-3.85(m, 1H), 3.63-3.47 (m, 5H), 3.43-3.25 (m, 3H), 3.25-3.11 (m, 2H), 2.33(s, 3H), 2.22 (t, J=15.2 Hz, 2H), 1.85-1.61 (m, 2H); LCMS (m/z): 452.2[M+H]⁺

Compound 284(S)-6-(1-acetylazetidin-3-yl)amino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide

Step 1: tert-butyl (1-acetylazetidin-3-yl)carbamate

To a solution of tert-butyl azetidin-3-ylcarbamate (100 g, 0.58 mol) andEt₃N (88 g, 0.87 mol) in DCM (1500 mL) was added Ac₂O (59.6 g, 0.88 mol)dropwise at 0° C. The mixture was then stirred at 0° C. for 2 h, atwhich time TLC showed the completion of the reaction. The reaction wasquenched by addition of water (1000 mL) and then stirred at 20° C. for30 min. The organic phase was separated, dried (Na₂SO₄) and concentratedto give the crude product. Seven batches were run in parallel andproduced a combined crude product weight of 530 g. This crude was usedin next step without the further purification. LCMS (m/z): 215.1 (M+1).

Step 2: 1-(3-aminoazetidin-1-yl)ethanone

To a solution of tert-butyl (1-acetylazetidin-3-yl)carbamate (250 g) inMeOH (1000 mL) was slowly added HCl/MeOH (4M, 300 mL) at 0° C. Afteraddition, the mixture was stirred at 0° C. for 6 h. The mixture was thenconcentrated under reduced pressure to give the crude product as a whitesolid. Two batches were run in parallel and produced a combined crudeproduct weight of 186 g. This crude was used in next step without thefurther purification. ¹H NMR (400 MHz, DMSO-d₆) δ 4.58-4.49 (m, 1H),4.35-4.19 (m, 2H), 4.19-4.08 (m, 1H), 3.97 (dd, J=4.2, 11.2 Hz, 1H),1.83 (s, 3H); LCMS (m/z): 115.1 (M+1).

Step 3: 6-chloropyrimidine-4-carbonyl chloride

A stirred mixture of 6-hydroxypyrimidine-4-carboxylic acid (75 g, 0.54mol) in EtOAc (300 mL) had oxalyl dichloride (226 g, 1.79 mol) droppedslowly to maintain the temperature below 30° C. After addition, themixture was stirred at 20° C. for 30 min and then DMF (2 mL) was addedto the mixture. The mixture was then stirred at 80° C. for 16 hours andconcentrated to give the crude product as a black solid. Sixteen batcheswere run in parallel and produced a combined crude product weight of1035 g. This crude was used directly in the next step.

Step 4:(S)-6-chloro-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide

To a stirred mixture of(S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (300 g, 1.46mol), and TEA (300 g, 3 mol) in DCM (4L) was added6-chloropyrimidine-4-carbonyl chloride (250 g in 2L of DCM) slowly at−60° C. over 1 h. After the addition was complete, the mixture was thenallowed to warm to 10° C. Stirring was continued for 1 h, at which timeTLC showed the reaction was completed. The reaction was quenched byaddition of water (2L). The organic phase was collected, dried (Na₂SO₄)and evaporated. The residue was purified by flash chromatography(EtOAc˜DCM:MeOH=10:1) to give the desired product as a pale yellowsolid. Four batches were run in parallel and produced a combined productweight of 970 g, yield: 49%. ¹H NMR (400 MHz, MeOD-d4) δ 8.73 (d, J=1.0Hz, 1H), 8.07 (d, J=1.2 Hz, 1H), 7.17-7.06 (m, 3H), 7.00 (d, J=7.2 Hz,1H), 5.51 (s, 1H), 4.12 (q, J=6.0 Hz, 1H), 3.74 (s, 2H), 3.64-3.53 (m,2H), 2.94 (q, J=5.6 Hz, 2H), 2.92-2.81 (m, 2H), 2.78-2.64 (m, 2H); LCMS(m/z): 347.2 [M+H]⁺

Step 5:(s)-6-((1-acetylazetidin-3-yl)amino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide

To a solution of(S)-6-chloro-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide(240 g, 0.69 mol) in i-PrOH (2.5L) was added 1-(3-aminoazetidin-1-yl)ethanone (120 g) and TEA (100 g). After addition, thesolution was heated at 60° C. for 16 h, at which time LCMS showedcompletion of the reaction. The mixture was concentrated and the residuewas purified by flash chromatography to give the crude product. Fourbatches were run in parallel and produced a combined crude productweight of 420 g, 90% purity. This crude was further purified onpreparative HPLC to give the title compound (330 g, >98% purity, freebase form). ¹H NMR (400 MHz, MeOD-d4) 8.27 (s, 1H), 7.12-6.98 (m, 5H),4.71 (s, 1H), 4.54 (m, 1H), 4.32 (m, 1H), 4.06 (m, 2H), 3.88 (m, 1H),3.70 (s, 2H), 3.53-3.50 (m, 2H), 2.91-2.83 (m, 4H), 2.65 (m, 2H), 1.88(s, 3H); LCMS (m/z): 425.2 [M+H]⁺

Step 6:(s)-6-((1-acetylazetidin-3-yl)amino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamidehydrochloride

The free base was dissolved in DCM (100 mL) and added dropwise to astirred and cooled solution of HCl (6N in EtOAc, 1 L) at −30° C.Stirring at −30° C. was continued for another 1 h and the resultingprecipitate was collected by filtration. The solid was washed with DCMand EtOAc, dried to give the HCl salt of the product (301 g, yield: 26%)as a white solid. ¹H NMR (400 MHz, D₂O) δ 8.65 (s, 1H), 7.30-7.19 (m,4H), 7.13 (d, J=7.5 Hz, 1H), 4.95-4.85 (m, 1H), 4.63-4.50 (m, 2H),4.41-4.28 (m, 3H), 4.22 (dd, J=4.8, 9.2 Hz, 1H), 3.97 (dd, J=4.6, 10.0Hz, 1H), 3.77 (dt, J=5.6, 11.3 Hz, 1H), 3.53-3.35 (m, 4H), 3.34-3.26 (m,1H), 3.22-3.04 (m, 2H), 1.87-1.79 (m, 3H); LCMS (m/z): 425.2 [M+H]⁺.

Biological Assays PRMT5 Biochemical Assay

General Materials.

S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), bicine, KCl,Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin (BSG), andTris(2-carboxyethyl)phosphine hydrochloride solution (TCEP) werepurchased from Sigma-Aldrich at the highest level of purity possible.³H-SAM was purchase from American Radiolabeled Chemicals with a specificactivity of 80 Ci/mmol. 384-well streptavidin Flashplates were purchasedfrom PerkinElmer.

Substrates. Peptide representative of human histone H4 residues 1-15 wassynthesized with a C-terminal linker-affinity tag motif and a C-terminalamide cap by 21^(st) Century Biochemicals. The peptide was highhigh-performance liquid chromatography (HPLC) purified to greater than95% purity and confirmed by liquid chromatography mass spectrometry(LC-MS). The sequence was Ac-SGRGKGGKGLGKGGA[K-Biot]-amide (SEQ IDNO.:3).

Molecular Biology:

Full-length human PRMT5 (NM_(—)006109.3) transcript variant 1 clone wasamplified from a fetal brain cDNA library, incorporating flanking 5′sequence encoding a FLAG tag (MDYKDDDDK) (SEQ ID NO.:4) fused directlyto Ala 2 of PRMT5. Full-length human MEP50 (NM_(—)024102) clone wasamplified from a human testis cDNA library incorporating a 5′ sequenceencoding a 6-histidine tag (MHHHHHH) (SEQ ID NO.:5) fused directly toArg 2 of MEP50. The amplified genes were sublconed into pENTR/D/TEV(Life Technologies) and subsequently transferred by Gateway™ attL×attRrecombination to pDEST8 baculvirus expression vector (LifeTechnologies).

Protein Expression.

Recombinant baculovirus and Baculovirus-Infected Insect Cells (BIIC)were generated according to Bac-to-Bac kit instructions (LifeTechnologies) and Wasilko, 2006, respectively. Protein over-expressionwas accomplished by infecting exponentially growing Spodopterafrugiperda (SF9) cell culture at 1.2×10⁶ cell/ml with a 5000 folddilution of BIIC stock. Infections were carried out at 27° C. for 72hours, harvested by centrifugation, and stored at −80° C. forpurification.

Protein Purification.

Expressed full-length human Flag-PRMT5/6His-MeP50 protein complex waspurified from cell paste by NiNTA agarose affinity chromatography aftera five hour equilibration of the resin with buffer containing 50 mMTris-HCL, pH 8.0, 25 mM NaCl, and 1 mM TCEP at 4° C., to minimize theadsorption of tubulin impurity by the resin. Flag-PRMT5/6His-MeP50 waseluted with 300 mM Imidazole in the same buffer. The purity of recoveredprotein was 87%. Reference: Wasilko, D. J. and S. E. Lee: “TIPS:titerless infected-cells preservation and scale-up” Bioprocess J., 5(2006), pp. 29-32.

Predicted Translations:

Flag-PRMT5 (SEQ ID NO.: 6) MDYKDDDDKA AMAVGGAGGS RVSSGRDLNC VPEIADTLGAVAKQGFDFLC MPVFHPRFKR EFIQEPAKNR PGPQTRSDLLLSGRDWNTLI VGKLSPWIRP DSKVEKIRRN SEAAMLQELNFGAYLGLPAF LLPLNQEDNT NLARVLTNHI HTGHHSSMFWMRVPLVAPED LRDDIIENAP TTHTEEYSGE EKTWMWWHNFRTLCDYSKRI AVALEIGADL PSNHVIDRWL GEPIKAAILPTSIFLTNKKG FPVLSKMHQR LIFRLLKLEV QFIITGTNHHSEKEFCSYLQ YLEYLSQNRP PPNAYELFAK GYEDYLQSPLQPLMDNLESQ TYEVFEKDPI KYSQYQQAIY KCLLDRVPEEEKDTNVQVLM VLGAGRGPLV NASLRAAKQA DRRIKLYAVEKNPNAVVTLE NWQFEEWGSQ VTVVSSDMRE WVAPEKADIIVSELLGSFAD NELSPECLDG AQHFLKDDGV SIPGEYTSFLAPISSSKLYN EVRACREKDR DPEAQFEMPY VVRLHNFHQLSAPQPCFTFS HPNRDPMIDN NRYCTLEFPV EVNTVLHGFAGYFETVLYQD ITLSIRPETH SPGMFSWFPI LFPIKQPITVREGQTICVRF WRCSNSKKVW YEWAVTAPVC SAIHNPTGRS YTIG L 6His-MEP50(SEQ ID NO.: 7) MHHHHHHRKE TPPPLVPPAA REWNLPPNAP ACMERQLEAARYRSDGALLL GASSLSGRCW AGSLWLFKDP CAAPNEGFCSAGVQTEAGVA DLTWVGERGI LVASDSGAVE LWELDENETLIVSKFCKYEH DDIVSTVSVL SSGTQAVSGS KDICIKVWDLAQQVVLSSYR AHAAQVTCVA ASPHKDSVFL SCSEDNRILLWDTRCPKPAS QIGCSAPGYL PTSLAWHPQQ SEVFVFGDENGTVSLVDTKS TSCVLSSAVH SQCVTGLVFS PHSVPFLASLSEDCSLAVLD SSLSELFRSQ AHRDFVRDAT WSPLNHSLLTTVGWDHQVVH HVVPTEPLPA PGPASVTE

General Procedure for PRMT5/MEP50 Enzyme Assays on Peptide Substrates.

The assays were all performed in a buffer consisting of 20 mM Bicine(pH=7.6), 1 mM TCEP, 0.005% BSG, and 0.002% Tween20, prepared on the dayof use. Compounds in 100% DMSO (1 ul) were spotted into a polypropylene384-well V-bottom plates (Greiner) using a Platemate Plus outfitted witha 384-channel head (Thermo Scientific). DMSO (1 ul) was added to Columns11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH,a known product and inhibitor of PRMT5/MEP50, was added to columns 11,12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul)containing the PRMT5/MEP50 enzyme and the peptide was added by MultidropCombi (Thermo-Fisher). The compounds were allowed to incubate withPRMT5/MEP50 for 30 min at 25 degrees Celsius, then a cocktail (10 ul)containing ³H-SAM was added to initiate the reaction (final volume=51ul). The final concentrations of the components were as follows:PRMT5/MEP50 was 4 nM, ³H-SAM was 75 nM, peptide was 40 nM, SAH in theminimum signal control wells was 100 uM, and the DMSO concentration was1%. The assays were stopped by the addition of non-radioactive SAM (10ul) to a final concentration of 600 uM, which dilutes the ³H-SAM to alevel where its incorporation into the peptide substrate is no longerdetectable. 50 ul of the reaction in the 384-well polypropylene platewas then transferred to a 384-well Flashplate and the biotinylatedpeptides were allowed to bind to the streptavidin surface for at least 1hour before being washed three times with 0.1% Tween20 in a BiotekELx405 plate washer. The plates were then read in a PerkinElmer TopCountplate reader to measure the quantity of ³H-labeled peptide bound to theFlashplate surface, measured as disintegrations per minute (dpm) oralternatively, referred to as counts per minute (cpm).

%  inhibition  calculation${\% \mspace{14mu} {inh}} = {100 - {\left( \frac{{dpm}_{cmpd} - {dpm}_{\min}}{{dpm}_{\max} - {dpm}_{\min}} \right) \times 100}}$

Where dpm=disintegrations per minute, cmpd=signal in assay well, and minand max are the respective minimum and maximum signal controls.

Four-parameter  IC50  fit$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{\left( {1 + {\left( \frac{X}{{IC}_{50}} \right){Hill}\mspace{14mu} {Coefficient}}} \right.}}$

Where top and bottom are the normally allowed to float, but may be fixedat 100 or 0 respectively in a 3-parameter fit. The Hill Coefficientnormally allowed to float but may also be fixed at 1 in a 3-parameterfit. Y is the % inhibition and X is the compound concentration.

Z-138 Methylation Assay

Z-138 suspension cells were purchased from ATCC (American Type CultureCollection, Manassas, Va.). RPMI/Glutamax medium,penicillin-streptomycin, heat inactivated fetal bovine serum, and D-PBSwere purchased from Life Technologies, Grand Island, N.Y., USA. Odysseyblocking buffer, 800CW goat anti-rabbit IgG (H+L) antibody, and LicorOdyssey infrared scanner were purchased from Licor Biosciences, Lincoln,Nebr., USA. Symmetric di-methyl arginine antibody was purchased from EMDMillipore, Billerica, Mass., USA. 16% Paraformaldehyde was purchasedfrom Electron Microscopy Sciences, Hatfield, Pa., USA.

Z-138 suspension cells were maintained in growth medium (RPMI 1640supplemented with 10% v/v heat inactivated fetal bovine serum and 100units/mL penicillin-streptomycin) and cultured at 37° C. under 5% CO₂

Cell Treatment, in Cell Western (ICW) for Detection of SymmetricDi-Methyl Arginine and DNA Content.

Z-138 cells were seeded in assay medium at a concentration of 50,000cells per mL to a 384-well cell culture plate with 50 μL per well.Compound (100 mL) from 384 well source plates was added directly to 384well cell plate. Plates were incubated at 37° C., 5% CO₂ for 96 hours.After four days of incubation, 40 μL of cells from incubated plates wereadded to poly-D-lysine coated 384 well culture plates (BD Biosciences356697). Plates were incubated at room temperature for 30 minutes thenincubated at 37° C., 5% CO₂ for 5 hours. After the incubation, 40 μL perwell of 8% paraformaldehyde in PBS (16% paraformaldahyde was diluted to8% in PBS) was added to each plate and incubated for 30 minutes. Plateswere transferred to a Biotek 405 plate washer and washed 5 times with100 μL per well of wash buffer (1×PBS with 0.1% Triton X-100 (v/v)).Next 30 μL per well of Odyssey blocking buffer were added to each plateand incubated 1 hour at room temperature. Blocking buffer was removedand 20 μL per well of primary antibody was added (symmetric di-methylarginine diluted 1:100 in Odyssey buffer with 0.1% Tween 20 (v/v)) andplates were incubated overnight (16 hours) at 4° C. Plates were washed 5times with 100 μL per well of wash buffer. Next 20 μL per well ofsecondary antibody was added (1:200 800CW goat anti-rabbit IgG (H+L)antibody, 1:1000 DRAQS (Biostatus limited) in Odyssey buffer with 0.1%Tween 20 (v/v)) and incubated for 1 hour at room temperature. The plateswere washed 5 times with 100 μL per well wash buffer then 1 time with100 μL per well of water. Plates were allowed to dry at room temperaturethen imaged on the Licor Odyssey machine which measures integratedintensity at 700 nm and 800 nm wavelengths. Both 700 and 800 channelswere scanned.

Calculations:

First, the ratio for each well was determined by:

$\left( \frac{{symmetric}\mspace{14mu} {di}\text{-}{methyl}\mspace{14mu} {Arginins}\mspace{14mu} 800\mspace{14mu} {nm}\mspace{14mu} {value}}{{DRAQ}\; 5\mspace{14mu} 700\mspace{14mu} {nm}\mspace{14mu} {value}} \right)$

Each plate included fourteen control wells of DMSO only treatment(minimum inhibition) as well as fourteen control wells for maximuminhibition treated with 3 μM of a reference compound (Background wells).The average of the ratio values for each control type was calculated andused to determine the percent inhibition for each test well in theplate. Reference compound was serially diluted three-fold in DMSO for atotal of nine test concentrations, beginning at 3 μM. Percent inhibitionwas determined and IC₅₀ curves were generated using triplicate wells perconcentration of compound.

${{Percent}\mspace{14mu} {Inhibition}} = {100 - \left( {\left( \frac{\left( {{Individual}\mspace{14mu} {Test}\mspace{14mu} {Sample}\mspace{14mu} {Ratio}} \right) - \left( {{Background}\mspace{14mu} {Avg}\mspace{14mu} {Ratio}} \right)}{\left( {{Minimum}\mspace{14mu} {Inhibition}\mspace{14mu} {Ratio}} \right) - \left( {{Background}\mspace{14mu} {Average}\mspace{14mu} {Ratio}} \right)} \right)*100} \right)}$

Z-138 Proliferation Assay

Z-138 suspension cells were purchased from ATCC (American Type CultureCollection, Manassas, Va.). RPMI/Glutamax medium,penicillin-streptomycin, heat inactivated fetal bovine serum werepurchased from Life Technologies, Grand Island, N.Y., USA. V-bottompolypropylene 384-well plates were purchased from Greiner Bio-One,Monroe, N.C., USA. Cell culture 384-well white opaque plates werepurchased from Perkin Elmer, Waltham, Mass., USA. Cell-Titer Glo® waspurchased from Promega Corporation, Madison, Wis., USA. SpectraMax M5plate reader was purchased from Molecular Devices LLC, Sunnyvale,Calif., USA.

Z-138 suspension cells were maintained in growth medium (RPMI 1640supplemented with 10% v/v heat inactivated fetal bovine serum andcultured at 37° C. under 5% CO₂. Under assay conditions, cells wereincubated in assay medium (RPMI 1640 supplemented with 10% v/v heatinactivated fetal bovine serum and 100 units/mL penicillin-streptomycin)at 37° C. under 5% CO₂.

For the assessment of the effect of compounds on the proliferation ofthe Z-138 cell line, exponentially growing cells were plated in 384-wellwhite opaque plates at a density of 10,000 cells/ml in a final volume of50 μl of assay medium. A compound source plate was prepared byperforming triplicate nine-point 3-fold serial dilutions in DMSO,beginning at 10 mM (final top concentration of compound in the assay was20 μM and the DMSO was 0.2%). A 100 mL aliquot from the compound stockplate was added to its respective well in the cell plate. The 100%inhibition control consisted of cells treated with 200 nM finalconcentration of staurosporine and the 0% inhibition control consistedof DMSO treated cells. After addition of compounds, assay plates wereincubated for 5 days at 37° C., 5% CO₂, relative humidity >90%. Cellviability was measured by quantitation of ATP present in the cellcultures, adding 35 μl of Cell Titer Glo® reagent to the cell plates.Luminescence was read in the SpectraMax M5 microplate reader. Theconcentration of compound inhibiting cell viability by 50% wasdetermined using a 4-parametric fit of the normalized dose responsecurves.

Results for certain compounds described herein are shown in Table 2.

TABLE 2 Biological Assay Results Cmpd No Biochemical IC₅₀ ICW EC₅₀Proliferation EC₅₀ 1 A A C 2 A A C 3 A A C 4 C — — 5 B B ** 6 C — — 7 C— — 8 A A C 9 A A C 10 A B C 11 A C — 12 B C ** 13 A A B 14 A B C 15 A BD 16 A A B 17 B B ** 18 B B D 19 A B D 20 A A B 21 B B ** 22 B B ** 23 BB ** 24 A B C 25 B C ** 26 B B D 27 C — — 28 A B D 29 A B C 30 A B C 31B B D 32 B B ** 33 C — — 34 A B D 35 A B D 36 A B D 37 A B ** 38 A B D39 B C ** 40 A A C 41 A A C 42 B C ** 43 B B C 44 A — B 45 A B — 46 C —— 47 B B — 48 B B — 49 B — — 50 B — — 51 B — — 52 B — — 53 B — — 54 A —— 55 A — — 56 B — — 57 B — — 58 A — — 59 A A B 60 B B C 61 B B — 62 A BD 63 A A B 64 A B C 65 A A C 66 A B C 67 B B D 68 A A C 69 B C ** 70 B C** 71 B B C 72 B C — 73 A A C 74 A A B 75 B B — 76 B B — 77 A B C 78 A AB 79 A A B 80 A A B 81 A B C 82 A A B 83 B B — 84 A A C 85 B B D 86 B C— 87 C — — 88 B B ** 89 B B ** 90 A B D 91 A A C 92 A A C 93 B C ** 94 AB D 95 B B C 96 A A C 97 A A C 98 A B C 99 A A C 100 A A C 101 A A D 102A A C 103 A A D 104 A A C 105 A A C 106 A A B 107 A A B 108 A A B 109 AA B 110 A A C 111 A B C 112 B C ** 113 A B D 114 A B D 115 A B ** 116 BB ** 117 B B ** 118 A B ** 119 A B C 120 A B C 121 A A B 122 A A B 123 AB C 124 A A A 125 A A B 126 A A C 127 A A C 128 A A C 129 A A C 130 A BD 131 A B C 132 A B C 133 A A C 134 A B D 135 A A D 136 A A C 137 A A C138 A B D 139 A A C 140 A A C 141 A A C 142 A A C 143 A A C 144 A A C145 A A C 146 A A C 147 A B D 148 B C ** 149 B C ** 150 B B ** 151 B B** 152 A A B 153 A A B 154 A B C 155 B C ** 156 A B C 157 B C ** 158 A —** 159 A B C 160 A B D 161 A A C 162 A A C 163 A A C 164 A A C 165 A B C166 A A B 167 A A B 168 A A B 169 A B C 170 B B ** 171 A B C 172 A A C173 A A C 174 A A C 175 A B C 176 A A C 177 A A C 178 A A C 179 A A C180 A B D 181 A B C 182 A A C 183 A A C 184 A A C 185 A B C 186 A A C187 A A B 188 A A A 189 A A B 190 A A B 191 A A B 192 A A B 193 A A B194 A B C 195 A B D 196 A A C 197 A A B 198 A A B 199 A A C 200 A B D201 A B C 202 A A C 203 A A B 204 A A B 205 A A B 206 A B C 207 A A B208 A A A 209 A A D 210 A A B 211 A A A 212 A A B 213 A A B 214 A A C215 A A A 216 A A B 217 A A B 218 A B D 219 A B D 220 A A B 221 A A B222 A B ** 223 A A C 224 A A B 225 A A A 226 A A B 227 A A B 228 A A B229 A A C 230 A A B 231 A A C 232 B B C 233 A B C 234 A A C 235 A B **236 A B C 237 A A C 238 A B D 239 A A C 240 B B ** 241 A B C 242 A B C243 A B C 244 A B C 245 A B D 246 A B C 247 A B C 248 A B C 249 A B D250 A A C 251 A A C 252 A B C 253 A B C 254 A A A 255 A A C 256 A A C257 A A C 258 A B D 259 A B ** 260 A B ** 261 A A C 262 A A B 263 A A C264 A A C 265 A B C 266 A A B 267 A A B 268 A A B 269 A A C 270 C — —271 A B C 272 A A C 273 A B C 274 A B C 275 B B C 276 A B C 277 A A —278 A A — 279 A A — 280 A A — 281 A B — 282 A A — 283 A A — 284 A A —285 B B — 286 A A — 287 A A — 288 A B — 289 B A — 290 A A — 291 A A —292 A A — 293 A A — 294 B B — 295 B B — 296 A B — 297 A A — 298 A A —299 A A — 300 A A — 301 A A — 302 A A — 303 A A — 304 A A — 305 B B —306 A — — 307 A — — 308 A — — 309 B — — 310 B — — 311 A — — 312 B — —313 — — — 314 A — — 315 A — — 316 A — — 317 A — — 318 A — — 319 A — —320 A — — 321 A — — 322 A — — 323 A — — 324 A — — 325 A — — 326 A — —327 B — — 328 C — — 329 A — — 330 A — — 331 A — — 332 A — — 333 A — —334 A — — 335 A — — For Table 2, “A” indicates an IC₅₀ or EC₅₀ < 0.100μM, “B” indicates an IC₅₀ or EC₅₀ of 0.101-1.000 μM, “C” indicates anIC₅₀ or EC₅₀ of 1.001-10.000 μM, “D” indicates an IC₅₀ or EC₅₀ of10.001-50 μM, and “E” indicates an IC₅₀ or EC₅₀ > 50 μM. “—” indicatesno data. “**” indicates an IC₅₀ or EC₅₀ > 20 μM.

OTHER EMBODIMENTS

The foregoing has been a description of certain non-limiting embodimentsof the invention. Those of ordinary skill in the art will appreciatethat various changes and modifications to this description may be madewithout departing from the spirit or scope of the present invention, asdefined in the following claims.

1. A compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein

represents a single or double bond; R¹ is hydrogen, R^(z), or—C(O)R^(z), wherein R^(z) is optionally substituted C₁₋₆ alkyl; L is—N(R)C(O)—, —C(O)N(R)—, —N(R)C(O)N(R)—, —N(R)C(O)O—, or —OC(O)N(R)—;each R is independently hydrogen or optionally substituted C₁₋₆aliphatic; Ar is a monocyclic or bicyclic aromatic ring having 0-4heteroatoms independently selected from nitrogen, oxygen, and sulfur,wherein Ar is substituted with 0, 1, 2, 3, 4, or 5 R^(y) groups, asvalency permits; each R^(y) is independently selected from the groupconsisting of halo, —CN, —NO₂, optionally substituted aliphatic,optionally substituted carbocyclyl, optionally substituted aryl,optionally substituted heterocyclyl, optionally substituted heteroaryl,—OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A), —C(O)OR^(A), —C(O)SR^(A),—C(O)N(R^(B))₂, —C(O)N(R^(B))N(R^(B))₂, —OC(O)R^(A), —OC(O)N(R^(B))₂,—NR^(B)C(O)R^(A), —NR^(B)C(O)N(R^(B))₂, —NR^(B)C(O)N(R^(B))N(R^(B))₂,—NR^(B)C(O)OR^(A), —SC(O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NOR^(A))R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(O)R^(A),—OS(O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), or —SO₂N(R^(B))₂; each R^(A)is independently selected from the group consisting of hydrogen,optionally substituted aliphatic, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl, andoptionally substituted heteroaryl; each R^(B) is independently selectedfrom the group consisting of hydrogen, optionally substituted aliphatic,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, and optionally substituted heteroaryl, ortwo R^(B) groups are taken together with their intervening atoms to forman optionally substituted heterocyclic ring; R⁵, R⁶, R⁷, and R⁸ areindependently hydrogen, halo, or optionally substituted aliphatic; eachR^(x) is independently selected from the group consisting of halo, —CN,optionally substituted aliphatic, —OR′, and —N(R″)₂; R′ is hydrogen oroptionally substituted aliphatic; each R″ is independently hydrogen oroptionally substituted aliphatic, or two R″ are taken together withtheir intervening atoms to form a heterocyclic ring; and n is 0, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10, as valency permits.
 2. The compound of claim1, wherein the compound is of Formula (I-a):

or a pharmaceutically acceptable salt thereof.
 3. The compound of claim1, wherein the compound is of Formula (I-b):

or a pharmaceutically acceptable salt thereof.
 4. The compound of claim1, wherein the compound is of Formula (I′):

or a pharmaceutically acceptable salt thereof.
 5. The compound of claim4, wherein the compound is of Formula (I′-a):

or a pharmaceutically acceptable salt thereof.
 6. The compound of claim4, wherein the compound is of Formula (I′-b):

or a pharmaceutically acceptable salt thereof.
 7. The compound of claim1, wherein L is —C(O)N(R)—, —NHC(O)NH—, or —OC(O)NH—. 8-9. (canceled)10. The compound of claim 1, wherein the compound is of Formula (II):

or a pharmaceutically acceptable salt thereof.
 11. The compound of claim10, wherein the compound is of Formula (II-a):

or a pharmaceutically acceptable salt thereof.
 12. The compound of claim10, wherein the compound is of Formula (II-b):

or a pharmaceutically acceptable salt thereof.
 13. The compound of claim1, wherein R¹ is hydrogen.
 14. The compound of claim 1, wherein n is 0,1, or
 2. 15-17. (canceled)
 18. The compound of claim 1, wherein Ar isheteroaryl.
 19. The compound of claim 18, wherein Ar is a 5- to6-membered heteroaryl having 1-3 heteroatoms independently selected fromnitrogen, oxygen, and sulfur.
 20. The compound of claim 19, wherein Aris pyridyl.
 21. (canceled)
 22. The compound of claim 1, wherein Ar issubstituted with 1 or 2 R^(y) groups.
 23. The compound of claim 22,wherein Ar is substituted with one R^(y) group. 24-32. (canceled) 33.The compound of claim 1, wherein the compound is of Formula (VI):

or a pharmaceutically acceptable salt thereof.
 34. The compound of claim1, wherein the compound is of Formula (VI-a):

or a pharmaceutically acceptable salt thereof.
 35. The compound of claim1, wherein the compound is of Formula (VI-b):

or a pharmaceutically acceptable salt thereof. 36-41. (canceled)
 42. Thecompound of claim 1, wherein the compound is of Formula (IX):

or a pharmaceutically acceptable salt thereof.
 43. The compound of claim1, wherein the compound is of Formula (IX-a):

or a pharmaceutically acceptable salt thereof.
 44. The compound of claim1, wherein the compound is of Formula (IX-b):

or a pharmaceutically acceptable salt thereof. 45-56. (canceled)
 57. Thecompound of claim 1, wherein the compound is of Formula (XV):

or a pharmaceutically acceptable salt thereof.
 58. (canceled)
 59. Thecompound of claim 1, wherein the compound is of Formula (XVII):

or a pharmaceutically acceptable salt thereof.
 60. (canceled)
 61. Thecompound of claim 1, wherein the compound is of Formula (XV-a):

or a pharmaceutically acceptable salt thereof.
 62. (canceled)
 63. Thecompound of claim 1, wherein the compound is of Formula (XVII-a):

or a pharmaceutically acceptable salt thereof. 64-82. (canceled)
 83. Thecompound of claim 1, wherein at least one R^(y) is —N(R^(B))₂. 84-86.(canceled)
 87. The compound of claim 83, wherein at least one R^(y) is—NHR^(B).
 88. The compound of claim 87, wherein R^(B) is optionallysubstituted heterocyclyl. 89-106. (canceled)
 107. The compound of claim1, wherein Ar is selected from the group consisting of:


108. The compound of claim 1, wherein the compound is selected from thegroup consisting of:


109. A pharmaceutical composition comprising a compound of claim 1, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable excipient.
 110. A kit or packaged pharmaceutical comprising acompound of claim 1 and instructions for use thereof.
 111. A method ofinhibiting PRMT5 comprising contacting a cell with an effective amountof a compound of claim 1 or a pharmaceutically acceptable salt thereof.112. A method of altering gene expression comprising contacting a cellwith an effective amount of a compound of claim 1 or a pharmaceuticallyacceptable salt thereof.
 113. A method of altering transcriptioncomprising contacting a cell with an effective amount of a compound ofclaim 1 or a pharmaceutically acceptable salt thereof.
 114. The methodof claim 1, wherein the cell is in vitro.
 115. The method of claim 1,wherein the cell is in a subject.
 116. A method of treating orpreventing a PRMT5-mediated disorder, comprising administering to asubject in need thereof a therapeutically effective amount of a compoundof claim 1, or a pharmaceutically acceptable salt thereof.
 117. Themethod of claim 116, wherein the disorder is a proliferative disorder, ametabolic disorder, or a blood disorder.
 118. The method of claim 117,wherein the proliferative disorder is cancer.
 119. The method of claim118, wherein the cancer is hematopoietic cancer, lung cancer, prostatecancer, melanoma, or pancreatic cancer.
 120. (canceled)
 121. The methodof claim 118, wherein the metabolic disorder is diabetes or obesity.122-123. (canceled)
 124. The method of claim 118, wherein the blooddisorder is a hemoglobinopathy.
 125. The method of claim 124, whereinthe blood disorder is sickle cell anemia or β-thalessemia. 126.(canceled)